Bacillus velezensis as well as separation method and application thereof

Abstract

The invention relates to the technical field of agricultural microbial agents, in particular to bacillus velezensis as well as a separation method and application thereof. The bacillus velezensis is preserved in the China General Microbiological Culture Collection Center, the preservation date is May 17, 2024, the preservation number is CGMCC No.30687, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the postcode is 100101, and the bacillus velezensis is classified and named as bacillus velezensis. The bacillus velezensis separated from the soil of the Ningboo garden of the Academy of Agricultural Sciences in Yitai City of Shandong Province or the fermentation inoculant thereof can be simultaneously used as a preparation raw material of a growth promoting agent, a yield increasing agent, a soil conditioner and a bio-organic fertilizer, and has multiple application scenes.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural microbial agents, and in particular to a strain of Bacillus velezinus and an isolation method and application thereof. Background Art

[0002] Protected cultivation is currently an important form of vegetable supply in my country, especially in Shandong Province, where intensive cultivation in greenhouses has promoted the increase of vegetable yields and year-round production and supply. However, due to limited land resources, most growers adopt a continuous cropping model without fallowing for year-round planting, especially for leafy vegetables with low economic value, which are planted continuously. This planting model has brought a serious burden to the soil in the greenhouse facilities, which can easily lead to soil continuous cropping obstacles. In addition, the evaporation of protected land is large, and there is a lack of rainwater washing. The degree of land salinization is getting worse, further exacerbating the agricultural ecological environment problems.

[0003] In recent years, with the progress of life sciences and the deepening of sustainable development models, the use of microbial agents and green inputs to continuously improve and repair agricultural soil has become an eco-friendly agricultural measure. The application of microbial agents can enhance the tolerance of plants to salinization environments, enhance plant resistance, and improve the soil ecological environment.

[0004] Bacillus velezensis belongs to the kingdom of bacteria, the phylum Firmicutes, the order Bacillus, the family Bacillaceae, and the genus Bacillus. It has strong stress resistance, including resistance to salt, acid and alkali, and high temperature. It can secrete and produce a variety of biologically active substances, such as enzymes (glucanase, protease, etc.), antimicrobial proteins, lipopeptide antibiotics (surfactant, iturin, fennectin, etc.), polyketide antibiotics, plant hormones (such as IAA, NH, etc.) and volatile compounds. At present, most Bacillus velezensis isolated from nature have only relatively single effects, such as: CN116622589B discloses a strain of Bacillus velezensis and its application in preventing and controlling fruit tree diseases. The deposited name of the Bacillus velezensis strain is Bacillus velezensis LS21, which has been deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC NO. 24447 and a deposit date of February 25, 2022; the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. However, the strain only has a good prevention and control effect on fruit tree diseases, and its uses in other application fields are unknown.

[0005] CN117165492B discloses a strain of Bacillus Velezii and its application, and its deposit number is CGMCC No. 22531. The above-mentioned Bacillus Velezii JNS-1 and its fermentation liquid can only be used to prepare medicines or fertilizers for preventing and controlling strawberry diseases, and the prevention and control of root diseases can solve the problems of serious plant death, chemical prevention and control easily pollutes groundwater, and unsatisfactory prevention effect. The use in other application fields is unknown.

[0006] CN114276957B is a biocontrol strain with the effect of preventing and treating a variety of plant pathogenic fungi and its application, which is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, with a deposit date of September 13, 2021, a deposit number of CGMCC No.23398, and a classification name: Bacillus velezensis. The strain in this technical solution can effectively prevent and treat watermelon wilt, pepper blight, strawberry root rot, cucumber root rot, tomato damping-off, and potato blight. This strain can only be used in the field of preventing and treating plant diseases, and its uses in other fields are unknown.

[0007] Based on the above-mentioned prior art, the Bacillus Velezii in the prior art has the technical problem that it can only prevent and treat plant diseases and has a narrow application field. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a strain of Bacillus velezensis, which is deposited in the General Microbiological Center of China Microorganism Culture Collection Committee (CGMCC), with a deposit date of May 17, 2024, a deposit number of CGMCC No. 30687, a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, zip code 100101, and a classification name of Bacillus velezensis; The bacterial cell characteristics of the Velez Bacillus are: wrinkled surface, irregular edges, and the bacterial cell morphology observed under an optical microscope is rod-shaped, single, Gram-positive, and has spores. Further, the gene sequence of the Velez Bacillus is shown in SEQ ID NO.1.

[0009] The present invention provides a method for isolating the above-mentioned Bacillus Velezii, comprising the following steps: Step 1, select the source of strains: select soil from Agricultural Expo Park of Yantai Academy of Agricultural Sciences, Shandong Province; Step 2, initial screening of strains: using gradient dilution plate method to separate strains and obtain initial screening colonies; Step 3, phosphate solubilization screening: observe the colonies initially screened in step 2 under a microscope, pick the strains with rod-shaped or slightly curved bodies, inoculate them into a phosphate solubilization medium and culture them at a constant temperature to perform the initial phosphate solubilization screening; pick the strains with similar bacterial characteristics to those of Bacillus Velezii, inoculate them into a new phosphate solubilization medium again, and culture them at a constant temperature again to obtain colonies with phosphate solubilization circles after phosphate solubilization screening; Step 4, confrontation culture screening: pick the strains from the colonies with phosphate-solubilizing rings, inoculate them into a culture medium containing strawberry root rot pathogens for antagonistic culture, and select strains with antagonistic effects; Step 5, strain separation and purification: select the strain with antagonistic effect with an antibacterial diameter of 0.9-1.2 cm in step 4, purify and culture it using LB medium, pick a single colony and preserve it as the original Bacillus Velezii; Step 6, gene sequencing: extracting the genomic DNA of the original Bacillus velezensis in step 5, sequencing the gyrA sequence gene, and its gene sequence is shown in SEQ ID NO.1; constructing a phylogenetic tree, and when the homology with the type species of Bacillus velezensis is 100%, the original Bacillus velezensis is determined to be the Bacillus velezensis.

[0010] Furthermore, the soil in step 1 comes from garden soil with rich plant diversity, and the soil layer thickness is selected to be ≤20 cm.

[0011] Furthermore, the specific operation steps of the gradient dilution plate method in step 2 are: Step 2-1, take the soil in step 1 and put it into a container filled with sterile water and shake it for the first time; Step 2-2, under sterile conditions, take the middle and upper layer of the liquid after shaking and put it into a sterile centrifuge tube filled with sterile water for one shaking; take the middle and upper layer of the liquid after shaking once and put it into a new sterile centrifuge tube filled with sterile water for a second shaking; repeat the above operation, dilute step by step, and shake seven times in total; Step 2-3: Take the liquid after the fifth, sixth, and seventh shaking in step 2-2 and inoculate it into the solid LB medium respectively, use a sterilized coating rod to spread it until the surface of the medium is dry, cover the culture dish with a lid, and culture in a constant temperature dark environment to obtain the primary screening colonies.

[0012] Furthermore, in step 2-1, the mass ratio of the soil to the sterile water in the container is 1:9, and the initial shaking time is 10 minutes.

[0013] Furthermore, the mass ratio of the liquid after shaking in step 2-2 to the sterile water in the sterilized centrifuge tube is 1:9, and the shaking time from one shaking to seven shaking is 2 minutes.

[0014] Furthermore, 100 μl of the liquid after shaking in step 2-3 was taken and inoculated into solid LB medium respectively.

[0015] Furthermore, the components of the solid LB medium in step 2-3 are 10 g / L tryptone, 5 g / L yeast extract, 15 g / L sodium chloride, and 18 g / L agar.

[0016] Furthermore, in step 2-3, the constant temperature is 28° C. and the incubation time is 48 h.

[0017] Furthermore, the components of the phosphate-dissolving medium in step 3 are 10 g / L glucose, 0.5 g / L magnesium sulfate, 0.2 g / L potassium chloride, 5 g / L calcium phosphate, 0.1 g / L ammonium sulfate, and 18 g / L agar.

[0018] Furthermore, in step 3, the constant temperature was 28° C. and the incubation time was 48 h.

[0019] Furthermore, the composition of the culture medium containing strawberry root rot bacteria in step 4 is 200 g / L peeled potatoes, 16 g / L glucose, and 18 g / L agar.

[0020] Furthermore, in step 4, the strawberry root rot pathogen is inoculated in the middle of the culture medium, and the strain with phosphate-solubilizing ring is evenly inoculated around the strawberry root rot pathogen. The culture temperature is 28° C. and the culture time is 48 hours.

[0021] Furthermore, the above strains or pathogens are all spot-planted on the surface of the culture medium using a sterile inoculation loop using a spot-planting method.

[0022] Furthermore, the above culture media were all operated using 90 mm culture dishes.

[0023] The present invention also provides a bacterial agent, which is the above-mentioned Bacillus Velez subtilis, or includes the above-mentioned Bacillus Velez subtilis.

[0024] The present invention also provides a fermentation agent, which is prepared by fermenting the above-mentioned Bacillus Velezii, and the fermentation steps are: Step 1, prepare fermentation medium: corn flour 20-22.5g / L, glucose 2.5-5g / L, soybean meal 5-7.5g / L, magnesium sulfate 0.25-0.5g / L, potassium dihydrogen phosphate 1-2g / L, potassium monohydrogen phosphate 0.5-1g / L; Step 2, inoculating the above-mentioned Bacillus Velez subtilis into the fermentation medium in step 1, with the inoculation amount being 1-10% of the total volume of the fermentation medium, and fermenting at 28-30° C. for 48 hours; Step 3: Filter the fermented bacterial liquid to obtain the fermentation agent.

[0025] Furthermore, the fermentation bacteria agent contains greater than or equal to 80% of Bacillus velez spores.

[0026] The present invention also provides a microbial agent for inhibiting pathogens, wherein the microbial agent comprises one or more of the above-mentioned Bacillus Velezii, or the above-mentioned agent, or the above-mentioned fermentation agent.

[0027] The present invention also provides a growth-promoting agent, which includes one or more of the above-mentioned Bacillus Velezii, or the above-mentioned bacterial agent, or the above-mentioned fermentation bacterial agent.

[0028] Furthermore, the growth-promoting agent is used for promoting the growth of plant seeds.

[0029] Furthermore, the growth promoter increases the growth rate of the plant seeds by 8.6% or more.

[0030] The present invention also provides a yield-increasing agent, which comprises one or more of the above-mentioned Bacillus Velez subtilis, the above-mentioned bacterial agent, or the above-mentioned fermentation bacterial agent.

[0031] Furthermore, the yield-increasing agent is used to increase vegetable yield.

[0032] Furthermore, the yield-increasing agent increases the yield of the vegetables by 6.02% or more.

[0033] The present invention also provides a soil conditioner, which comprises one or more of the above-mentioned Bacillus Velez subtilis, the above-mentioned bacterial agent, or the above-mentioned fermentation bacterial agent.

[0034] Furthermore, the soil conditioner can simultaneously improve the transplant survival rate and yield of plants, and has the performance of strong phosphorus solubilization, auxin production, and improving the salt-alkali tolerance of plants.

[0035] Furthermore, the use of the soil conditioner in plant transplanting can increase the transplanting survival rate by 6.3% or more and the yield by 5.5% or more.

[0036] The present invention also provides a bio-organic fertilizer, which comprises one or more of the above-mentioned Bacillus Velez subtilis, the above-mentioned bacterial agent, or the above-mentioned fermentation bacterial agent.

[0037] Furthermore, the bio-organic fertilizer is used for composting, fermentation or colonization.

[0038] Furthermore, the bio-organic fertilizer also includes biochar and Cnidium monnieri.

[0039] Furthermore, the particle size of the biochar is 20 mesh.

[0040] Furthermore, the added amount of the biochar is 5-10wt% of the entire bio-organic fertilizer.

[0041] Furthermore, the added amount of the Cnidium monnieri is 0.5-1wt% of the entire bio-organic fertilizer.

[0042] Furthermore, after the shelf life of the bio-organic fertilizer is 6 months, the number of live bacteria in the bio-organic fertilizer is detected to be ≥ 40 million / g. The beneficial effects of the present invention are: 1. The Velez subtilis or its fermented bacterial agent separated from the soil of the Agricultural Expo Park of the Yantai Academy of Agricultural Sciences in Shandong Province by a specific separation method can be used as a growth promoter, a yield increaser, a soil conditioner, and a raw material for preparing biological organic fertilizers. It has multiple application scenarios, breaking the existing application scope of Velez subtilis that can only inhibit pathogens, and can significantly improve the survival rate of vegetables after transplanting, promote plant germination and growth, and improve the salt and alkali tolerance of plants. 2. When the Bacillus Velez isolated by the present invention is used to produce a promoter, it is used to promote the growth of plant seeds. The promoter increases the growth rate of the plant seeds by 8.6% or more because the Bacillus Velez isolated by the present invention can produce 5wt% higher indoleacetic acid than the existing strains of Bacillus Velez, and the substance has the effect of promoting cell division and plant growth, and can better promote the germination of plant seeds; 3. The present invention ferments the Bacillus Velezius isolated by the present invention by a specific fermentation medium to obtain a fermentation agent. The content of indoleacetic acid in the fermentation agent prepared by the present invention is 15 mg / L higher than that obtained by using a common LB liquid culture medium. A higher concentration of indoleacetic acid can promote faster germination or growth of plant seeds. 4. The yield-increasing agent prepared by the present invention has a certain promoting effect on vegetable yield, which is 6.02% or more higher than the control. The yield-increasing agent prepared by using the isolated Bacillus Velezii of the present invention can play a buffering role on the soil environment. First, the strain is in a spore state and can be awakened and germinated after application, so it can better adapt to the soil environment. Moreover, as it is separated from the garden soil with rich soil microbial diversity, it can quickly adapt to the indigenous soil flora, establish a microecology, occupy an ecological niche, promote the diversity of soil bacterial communities, and cause certain changes in the bacterial communities. After that, active substances that are beneficial to the growth of vegetables are produced during the planting process, promoting the growth of vegetables. At the same time, the metabolites already in the yield-increasing agent can also provide nutrition and defense for vegetables, helping vegetables to adapt to the environment and resist adverse environments and pathogens. 5. The soil conditioner prepared by the present invention has the effect of increasing the yield while improving the transplant survival rate, that is, it can significantly affect the soil bacterial colony, promote the changes in the indigenous microbial colony, especially the changes in the bacterial community. In addition to the activity of the metabolically active substances in the soil conditioner itself, it can also promote other bacterial communities to have a positive effect on plants, promote plant growth and salt tolerance, thereby simultaneously improving the transplant survival rate and yield of plants; 6. The biological organic fertilizer prepared by the present invention can better reduce the biological effectiveness of heavy metals, passivate heavy metals, and reduce their harm to the environment. Under the cooperation of the fermentation agent prepared by the Bacillus Velezii isolated by the present invention and the biochar and Cnidium monnieri, on the one hand, it has a certain promoting effect on the degradation of malodorous gases such as nitrogen oxides and sulfides, and on the other hand, the biological organic fertilizer prepared by the present invention has a certain repellency to pests. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the primary screening colony map in Example 1 of the present invention; Figure 2 This is the initial phosphorus solution screening diagram in Example 1 of the present invention; Figure 3 This is a diagram of the confrontation culture screening in Example 1 of the present invention; Figure 4 This is a diagram of the bacterial morphology in Example 1 of the present invention; Figure 5 This is a bacterial colony morphology diagram in Example 1 of the present invention; Figure 6 It is a homology comparison diagram in Example 1 of the present invention; Figure 7 This is a diagram showing the analysis of the liquid quality detection results in Example 2 of the present invention; Figure 8 This is a comparison chart of the Shannon index of the experimental group and the blank group in Example 4 of the present invention; Fig. 9 This is a comparison chart of the Shannon index of the experimental group, control group and blank group in Example 5 of the present invention. DETAILED DESCRIPTION Example

[0045] This embodiment provides a method for isolating Bacillus Velezii, comprising the following steps: Step 1, select the source of strains: select soil from Agricultural Expo Park of Yantai Academy of Agricultural Sciences, Shandong Province; Among them, the soil comes from the garden soil with rich plant diversity, and the soil layer thickness is selected to be 20cm; Step 2, initial screening of strains: using gradient dilution plate method to separate strains and obtain initial screening colonies; Among them, the specific operation steps of the gradient dilution plate method are: Step 2-1, take 10g of soil from step 1 and put it into a container containing 90mL of sterile water and shake it for the first time for 10min; Step 2-2, under sterile conditions, take 1 mL of the middle and upper layer liquid after shaking and put it into a sterile centrifuge tube filled with 9 mL of sterile water for shaking for 2 minutes; take 1 mL of the middle and upper layer liquid after shaking again and put it into a new sterile centrifuge tube filled with 9 mL of sterile water for a second shaking for 2 minutes; repeat the above operation, dilute step by step, and shake seven times in total; Step 2-3: Take 100 μl of the liquid after the fifth, sixth, and seventh shaking in step 2-2 and inoculate it into the solid LB medium respectively. Use a sterilized coating rod to spread it until the surface of the medium is dry. Cover the culture dish with a lid and culture it in a dark environment at 28°C for 48 hours to obtain the initial screening colonies. Figure 1 As shown; The components of the solid LB medium are 10 g / L tryptone, 5 g / L yeast extract, 15 g / L sodium chloride, and 18 g / L agar. Different from the prior art, the concentration of sodium oxide in this embodiment is adjusted from 10 g / L to 15 g / L, in order to increase the selection pressure of salt concentration and screen strains resistant to higher salt concentrations. Step 3, phosphate solubilization screening: Observe the colonies screened in step 2 under a microscope, pick out the strains with rod-shaped or slightly curved bodies, inoculate them into phosphate solubilization medium and culture them at a constant temperature for the initial phosphate solubilization screening. Figure 2 As shown; strains with bacterial characteristics similar to those of Bacillus Velez are selected, inoculated again on a new phosphate-solubilizing medium, and cultured again at a constant temperature to obtain colonies with phosphate-solubilizing circles after phosphate-solubilizing screening; The composition of the phosphate-solubilizing medium is glucose 10g / L, magnesium sulfate 0.5g / L, potassium chloride 0.2g / L, calcium phosphate 5g / L, ammonium sulfate 0.1g / L, and agar 18g / L; there are tiny insoluble calcium phosphates in the phosphate-solubilizing medium, which decompose and release soluble phosphates when encountering acid, and the insoluble matter dissolves to form a phosphate-solubilizing circle, which proves that the strain has the function of phosphate-solubilizing; The above constant temperature is 28°C and the culture time is 48h.

[0046] Step 4, confrontation culture screening: pick the strains from the colonies with phosphate-solubilizing rings, inoculate them into a culture medium containing strawberry root rot pathogens for antagonistic culture, and select strains with antagonistic effects; The composition of the culture medium containing strawberry root rot bacteria is 200g / L peeled potato, 16g / L glucose, and 18g / L agar. The strawberry root rot bacteria are inoculated in the middle of the culture medium, and the strains with phosphate-solubilizing rings are evenly inoculated around the strawberry root rot bacteria, that is, at the four vertices in the positive direction, such as Figure 3 As shown, the culture temperature was 28°C and the culture time was 48 h; Step 5, strain separation and purification: select the strain with the largest antibacterial diameter in step 4, that is, select the strain with an antagonistic effect of 1.2 cm in diameter, use the solid LB medium with the same components as in step 2 for purification and culture, pick a single colony and save it as the original Bacillus Velezii; its bacterial morphology is as follows Figure 4 The colony morphology is shown in Figure 5 As shown; wherein, the bacterial cell characteristics of the Velez Bacillus are: wrinkled surface, irregular edges, and the bacterial cell morphology observed under an optical microscope is rod-shaped, single, Gram-positive, and has spores; Step 6, gene sequencing: The genomic DNA of the original Bacillus velezensis in step 5 is sequenced based on the gyrA sequence gene, and its gene sequence is shown in SEQ ID NO.1; a phylogenetic tree is constructed, and when the homology with the type species of Bacillus velezensis is 100%, Figure 6 As shown, the original Bacillus velezensis was determined to be the Bacillus velezensis of the present invention, and was named HL01; Figure 6 In the middle, Bacillus velezensis strain AMR25: Bacillus velezensis strain AMR25; Bacillus velezensis strain LF01: Bacillus velezensis strain LF01; Bacillus velezensis strain JBCS608: Bacillus velezensis strain JBCS608; Bacillus amyloliquefaciens strain TL106: Bacillus amyloliquefaciens strain TL106; Bacillus thuringiensis ATCC 10792: Bacillus thuringiensis strain ATCC 10792; Bacillus altitudinis strain GR-8: Bacillus altitudinis strain GR-8; Bacillus subtilis str.168: Bacillus subtilis; Bacillus amyloliquefaciens strain GKT04: Bacillus amyloliquefaciens; Bacillus siamensis KCTC 13613: Bacillus siamensis strain KCTC13613.

[0047] In this embodiment, the above strains or pathogens are all implanted on the surface of the culture medium using a sterile inoculation loop by the implantation method; and the above culture medium is operated using a 90 mm culture dish.

[0048] Finally, the strain obtained in this example was preserved. The Bacillus velezensis (HL01) was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration (CGMCC). The deposit date was May 17, 2024. The deposit number was CGMCC No. 30687. The deposit address was No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, and the classification name was Bacillus velezensis.

[0049] Example 2 This embodiment provides a growth promoter, wherein the growth promoter is to promote the growth of cucumber seeds using the Bacillus Velezii (HL01) separated in Example 1, and the specific steps are as follows: The Velezella isolated in Example 1 was inoculated into LB liquid culture medium with a sterile inoculation loop, and cultured at 28° C. and 180 rpm / min for 48 hours. The culture solution was filtered and diluted 1000 times. The diluted culture solution was used to soak cucumber seeds for 1 hour, which was the Example group; At the same time, the strain of Bacillus Velezii CGMCC No. 17842 in the prior art was used as a control group, and the clear water culture was used as a blank group, and the above operations were performed simultaneously.

[0050] Then, the three groups of cucumber seeds were placed in culture dishes (90 mm), with 10 seeds in each dish. Each treatment was repeated 3 times. Pre-moistened filter paper was placed in each dish to keep it moist. The dish was covered with a lid and cultured at room temperature for 3 days.

[0051] The growth promotion results of cucumber seeds are shown in Table 1. It can be seen that the Velez subtilis isolated by the present invention has a significant increase compared with the control group and the blank group, with the highest increase of 8.6%.

[0052] Table 1 Growth promotion results of cucumber seeds (unit: cm)

[0053] The components of the culture fluid separated by the present invention were qualitatively analyzed by liquid chromatography-mass spectrometry: (1) Sample preparation: The culture broth after the above fermentation was centrifuged at 8000 rpm / min for 5 min, and the supernatant was filtered using a 0.22 μm filter membrane to obtain a filtrate; (2) Liquid chromatography separation: A C18 column was used, and the mobile phase was water and acetonitrile / methanol for gradient elution of the filtrate; (3) Mass spectrometry detection: Electrospray positive ionization is used for scanning to obtain mass spectrometry data, such as Figure 7 As shown, the peak data indicates that it contains a higher relative content of indoleacetic acid, which is 5wt% higher than that of the strain of Bacillus Velezii CGMCC No.17842. This substance has the effect of promoting cell division and plant growth. Therefore, the Bacillus Velezii isolated by the present invention contains more indoleacetic acid in the culture solution after fermentation compared with the existing Bacillus Velezii, which leads to the effect of promoting the growth of cucumber seed roots.

[0054] Example 3 This embodiment provides a fermentation agent, which is prepared by fermenting the Bacillus Velezii isolated in Example 1, and the fermentation steps are: Step 1, prepare the specific fermentation medium of this embodiment: corn flour 20-22.5g / L, glucose 2.5-5g / L, soybean meal powder 5-7.5g / L, magnesium sulfate 0.25-0.5g / L, potassium dihydrogen phosphate 1-2g / L, potassium monohydrogen phosphate 0.5-1g / L; specifically, in this embodiment, the formula of 2L culture medium is as follows: corn flour 45g, glucose 9g, soybean meal powder 12g, magnesium sulfate 0.8g, potassium dihydrogen phosphate 3g, potassium monohydrogen phosphate 1g; Step 2, inoculating the above-mentioned Bacillus Velez subtilis into the fermentation medium in step 1, with an inoculation amount of 3% (volume ratio), and fermenting at 28° C. for 48 hours, which is a fermentation example group; At the same time, the above-mentioned Bacillus Velezii was inoculated into ordinary LB liquid culture medium with an inoculation amount of 3% (volume ratio) and fermented at 28°C for 48 hours as the fermentation control group. Each group was repeated 3 times. The fermentation medium of this embodiment is different from the medium of pure culture. The nutrients of the medium of pure culture are relatively simple, and more carbon sources and nitrogen sources are directly utilized. The specific fermentation medium of this embodiment adds crude raw materials, and the direct carbon and nitrogen sources are relatively few. Bacillus Velezii needs to undergo more life activities to decompose the substrate before it can be utilized. The fermentation process is accompanied by the production of spores. For a total of 48 hours, 80% of the spores are formed, and the pH shows a trend of first decreasing, then recovering, and finally slightly increasing. Among them, 24 hours after inoculation, a small amount of fermentation liquid was taken and observed under an optical microscope. It was found that the number of strains was increasing. Then samples were taken every hour to observe the formation of spores and the increase or decrease in the number of bacteria. The number of spores reached the maximum at around 48 hours. If some early-mature strains are continued to be cultured, autolysis may occur, accompanied by the risk of infection with miscellaneous bacteria, which may lead to phage infection and fermentation failure. Therefore, the time of spore formation is used as an indicator of fermentation cessation, so the specific fermentation time in this embodiment is set to 48 hours; Step 3: Filter the bacterial liquid of the fermentation example group and the fermentation control group after fermentation in step 2 to obtain the fermentation bacterial agent of this example and the control fermentation bacterial agent.

[0055] The fermentation bacteria of this example and the control fermentation bacteria were qualitatively analyzed by the same analysis method as in Example 2. It was found that the content of indoleacetic acid in the fermentation bacteria of this example was 15 mg / L higher than that in the control fermentation bacteria.

[0056] This proves that the Bacillus Velez isolated by the present invention can be used in a fermentation medium with specific components and proportions to obtain a fermentation agent that produces more indoleacetic acid, and can promote faster germination and growth of plant seeds during application.

[0057] Example 4 This embodiment provides a yield-increasing agent, which is a cucumber field test using the fermentation agent in Example 3 as the yield-increasing experimental group, and an equal amount of uninoculated ordinary LB liquid culture medium as the yield-increasing blank group, and each group is repeated 3 times.

[0058] Drip irrigation was carried out twice during the cucumber seedling stage and fruiting stage, with 1L per mu per time, drip irrigation with water; 30m 2 ; After applying twice, the 0-20cm soil of the cucumber test field was sampled. A 5-point sampling method was used. Five sampling points were taken for each treatment. 100g of soil was taken from each sampling point. After mixing, it was mailed to Shanghai Meiji Biomedical Technology Co., Ltd. for sequencing of soil bacterial diversity to analyze whether the yield-increasing agent had an effect on the soil microbial community; at the time of harvest, the yield data of the cucumber was statistically analyzed. The results are shown in Table 2: Table 2 Cucumber yield statistics (unit: kg)

[0059] It can be seen from Table 2 that the yield-increasing agent prepared in this example has a certain promoting effect on cucumber yield, which is 6.02% higher than that of the blank group.

[0060] Among them, the main steps of the determination of bacteria in the soil by Shanghai Meiji Biotechnology Co., Ltd. are as follows: first, the soil microbial genome is extracted. After the genomic DNA is extracted, the extracted genomic DNA is detected by 1% agarose gel electrophoresis. Then, the 16s rRNA V3-V4 region is amplified by PCR, and the PCR products are mixed and detected by 2% agarose gel electrophoresis, and purified using AMPure® PB beads (Pacifc Biosciences, CA, USA) magnetic beads. The PCR products are detected and quantified using the QuantiFluor-ST blue fluorescence quantification system (Promega), and then mixed in corresponding proportions according to the sequencing requirements of each sample. MiSeq / Novaseq library construction and sequencing are then performed, followed by bioinformatics analysis. The results are as follows. Figure 8 As shown by Figure 8 It can be seen that the Shannon index of the yield-increasing agent treatment was significantly increased compared with the blank group treatment, indicating that the soil bacterial diversity index of the experimental group was significantly improved.

[0061] After cucumber is transplanted, it faces the change of growth environment from plug tray to soil. Cucumber in seedling stage needs to resist many changes such as pathogens, soil structure and water and fertilizer in the transplanting area environment. The yield-increasing agent prepared by using the specific fermentation agent of the present invention can play a buffering role on the soil environment. First, the strain is in a spore state and can be awakened after application, germinate, and can better adapt to the soil environment. And as a garden soil with rich soil microbial diversity, it can quickly adapt to the soil indigenous flora, establish microecology, occupy ecological niche, promote the diversity of soil bacterial community, make bacterial community produce certain changes, and then produce active substances that are beneficial to cucumber growth in the field planting process, promote cucumber growth. At the same time, the metabolites already in the yield-increasing agent can also nourish and defend cucumber, help cucumber adapt to the environment, resist adverse environment and pathogens.

[0062] Therefore, the yield-increasing agent prepared by the specific fermentation bacteria of the present invention can be used to prepare a microbial preparation for promoting plant growth, especially for preparing a microbial preparation for promoting cucumber growth.

[0063] Example 5 The present embodiment provides a soil conditioner, which adopts the fermentation agent in Example 3, takes lettuce as the test object, selects a facility greenhouse for long-term continuous planting for the test, and is a transplanting experimental group. At the same time, an equal amount of CGMCC No.17842 Velez subtilis strain and an uninoculated ordinary LB liquid culture medium are inoculated as a transplanting control group and a transplanting blank group, and each group is repeated 3 times.

[0064] That is, the roots of the lettuces were dipped when they were transplanted. The bacterial agents in the transplanting experimental group, the transplanting control group and the transplanting blank group were filtered and diluted 400 times for root dipping.

[0065] The survival rate was calculated 10 days after transplanting and the results are shown in Table 3.

[0066] Table 3 Statistics of survival rate of lettuce after transplanting (unit: %)

[0067] It can be seen from the table that the survival rate of the soil conditioner prepared by using this embodiment is improved by 6.3% compared with the survival agent prepared by the existing Bacillus Velez subtilis, and is 10.9% higher than the blank group, that is, the blank group not inoculated with Bacillus Velez subtilis.

[0068] After the transplanting survived, the transplanting experimental group, the transplanting control group and the transplanting blank group were treated with drip irrigation of the inoculant. Each treatment was repeated 3 times, each plot was 30 square meters, and the treatment was drip irrigated twice. The yield of the lettuce was measured after harvesting, and soil samples were taken in the late growth stage of the lettuce for analysis of soil bacterial diversity. The sampling method was the same as that in Example 4.

[0069] Table 4 Statistics of lettuce production (unit: kg)

[0070] As can be seen from Table 4, the soil conditioner prepared in this embodiment not only has the effect of improving the transplant survival rate but also has the effect of increasing the yield, which is improved by 5.5% compared with the existing survival agent prepared by Bacillus Velez subtilis, and is 10.4% higher than the blank group, that is, the blank group not inoculated with Bacillus Velez subtilis.

[0071] The soil samples of the late growth stage of lettuce were sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for 16s rRNA sequencing. The sequencing process and method were the same as in Example 4. The results are as follows: Fig. 9 As shown, it can be seen from the figure that the Shannon index of the experimental group corresponding to the soil conditioner is significantly higher than that of the control group and the blank group, indicating that the Velez subsporins separated by the present invention can significantly improve the diversity of soil bacteria after being prepared into a soil conditioner and applied. Comprehensive survival rate and yield, as well as bacterial community diversity analysis, the Velez subsporins separated by the present invention can significantly affect the soil bacterial colony after application, promote the changes in the indigenous microbial colony, especially the changes in the bacterial community, in addition to the activity of the metabolically active substances in the soil conditioner itself, it can also promote other bacterial communities to have a positive effect on plants, promote the growth and salt tolerance of lettuce, thereby simultaneously improving the transplant survival rate and yield of lettuce.

[0072] Therefore, the Velez subspore separated and the fermentation agent prepared therefrom have the functions of promoting, dissolving phosphorus and improving the salt-alkali tolerance of plants, and can be used to prepare a soil conditioner for saline-alkali land.

[0073] Example 6 This embodiment provides a bio-organic fertilizer, which uses chicken manure as a raw material to prepare the bio-organic fertilizer containing the fermentation agent in Example 3.

[0074] Add straw or edible mushroom sticks (waste materials) to the chicken manure raw materials to adjust the carbon-nitrogen ratio of the initial composting raw materials to between (25-30):1, and the moisture content of the raw material pile to 60%, so that the effective live bacteria count of the strain is not less than 30 million / g at the end of fermentation.

[0075] In this embodiment, 6 kg of fermentation agent completely fermented in Example 3 was added to 1 cubic meter of chicken manure, and the number of viable bacteria was 5*10 8 cfu / L, in order to promote the passivation of heavy metals in chicken manure and reduce the secondary pollution released by chicken manure into the environment, 8wt% of 20-mesh biochar and 0.8wt% of Cnidium monnieri plant powder are added to each cubic meter of chicken manure to repel insects (such as flies, etc.). Chicken manure composting should be covered with breathable but impermeable fabric. The fermentation cycle is 20d in summer and 45d in winter. The germination index is used as the criterion for judging the completion of composting. If the germination index is greater than or equal to 80%, the composting is considered to be complete. This embodiment is an experiment conducted in July in summer, and the germination index is 81%. Therefore, the number of live bacteria in the biological organic fertilizer prepared in this embodiment is 400 million / g after a shelf life of 6 months, which is the biological organic fertilizer experimental group; At the same time, the strain of Bacillus Velezii CGMCC No.17842 in the prior art was used as a biological control group of organic fertilizer, and the above operations were performed simultaneously.

[0076] The bio-organic fertilizer experimental group prepared in the present embodiment is compared with the control group, and the low bioavailability of heavy metal Cr in chicken manure increases by 3.2%, and the high bioavailability decreases by 5.8%, wherein bioavailability refers to the ratio of the amount of chemicals that can be absorbed by the digestive tract after entering the human body, and finally reaches the blood or lymphatic tissue (i.e., enters the human body circulation) to the total intake, and is measured by animal experiments. The chicken manure bio-organic fertilizer prepared by the bio-organic fertilizer and biochar prepared in the present embodiment is described, and the bio-availability of heavy metals can be better reduced, the heavy metals can be passivated, and the harm to the environment can be reduced. It is because the Velez bacillus obtained by the present invention is separated and obtained in combination with specific fermentation conditions, which accelerates the rate at which the fermentation system animal and plant source waste is converted into other macromolecular compounds, and biochar can provide a better habitat for microbial agents, and can adsorb macromolecular substances to form a stable complex, which forms an obstacle to the effectiveness of heavy metals.

[0077] In this embodiment, Cnidium monnieri was added to chicken manure, and the number of flies trapped in the chicken manure with Cnidium monnieri was compared with that in the chicken manure without Cnidium monnieri and using the same amount of inoculant prepared by the strain of Bacillus Velezii of CGMCC No.17842 by hanging yellow sticky insect boards during the fermentation process. This shows that the biological organic fertilizer prepared in this embodiment needs to be prepared by the fermentation inoculant prepared by the Bacillus Velezii isolated in the present invention and cooperate with the biochar and Cnidium monnieri to achieve the corresponding technical effect, that is, on the one hand, it has a certain promoting effect on the degradation of malodorous gases such as nitrogen oxides and sulfides, and on the other hand, the biological organic fertilizer prepared in this embodiment has a certain repellency to pests.

[0078] In summary, the Bacillus Velez subtilis or fermentation agent separated from the soil of the Agricultural Expo Park of Yantai Academy of Agricultural Sciences in Shandong Province by a specific separation method in the present invention can be used as a growth promoter, a yield increaser, a soil conditioner, and a raw material for the preparation of biological organic fertilizers. It has multiple application scenarios, breaking the existing application scope of Bacillus Velez subtilis that can only be used to inhibit pathogens, and can significantly improve the survival rate of vegetables after transplanting, promote plant germination and growth, and improve the salt and alkali tolerance of plants.

[0079] It should be understood that the present invention is not limited to what has been described above and shown in the accompanying drawings, and that various modifications and changes may be made without departing from the scope thereof. The scope of the present invention is limited only by the appended claims.

Claims

1. A strain of Bacillus Velez, characterized in that: The Bacillus velezensis is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration Committee on May 17, 2024, with a deposit number of CGMCC No.30687, a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, zip code 100101, and a classification name of Bacillus velezensis; The bacterial characteristics of the Velez Bacillus are: wrinkled surface, irregular edges, and the bacterial morphology is rod-shaped and single when observed under an optical microscope. It is Gram-positive and has spores. The gene sequence of the Bacillus Velez is shown in SEQ ID NO.

1.

2. A method for isolating Bacillus Velezii according to claim 1, characterized in that: The steps include: Step 1, select the source of strains: select soil from Agricultural Expo Park of Yantai Academy of Agricultural Sciences, Shandong Province; Step 2, initial screening of strains: using gradient dilution plate method to separate strains and obtain initial screening colonies; Step 3, phosphate solubilization screening: observe the colonies initially screened in step 2 under a microscope, pick the strains with rod-shaped or slightly curved bodies, inoculate them into a phosphate solubilization medium and culture them at a constant temperature to perform the initial phosphate solubilization screening; pick the strains with similar bacterial characteristics to those of Bacillus Velezii, inoculate them into a new phosphate solubilization medium again, and culture them at a constant temperature again to obtain colonies with phosphate solubilization circles after phosphate solubilization screening; Step 4, confrontation culture screening: pick the strains from the colonies with phosphate-solubilizing rings, inoculate them into a culture medium containing strawberry root rot pathogens for antagonistic culture, and select strains with antagonistic effects; Step 5, strain separation and purification: select the strain with antagonistic effect with an antibacterial diameter of 0.9 cm-1.2 cm in step 4, purify and culture it using LB medium, pick a single colony and preserve it as the original Bacillus Velezii; Step 6, gene sequencing: The genomic DNA of the original Bacillus velezensis in step 5 is sequenced based on the gyrA sequence gene, and its gene sequence is shown in SEQ ID NO.1; a phylogenetic tree is constructed, and when the homology with the type species of Bacillus velezensis is 100%, the original Bacillus velezensis is determined to be the Bacillus velezensis.

3. The method for isolating Bacillus Velezii according to claim 2, characterized in that: The specific operation steps of the gradient dilution plate method in step 2 are as follows: Step 2-1, take the soil in step 1 and put it into a container filled with sterile water and shake it for the first time; Step 2-2, under sterile conditions, take the middle and upper layer of the liquid after shaking and put it into a sterile centrifuge tube filled with sterile water for one shaking; take the middle and upper layer of the liquid after shaking once and put it into a new sterile centrifuge tube filled with sterile water for a second shaking; repeat the above operation, dilute step by step, and shake seven times in total; Step 2-3, take the liquid after the fifth, sixth, and seventh shaking in step 2-2 and inoculate it into the solid LB medium respectively, use a sterilized coating rod to spread it until the surface of the medium is dry, cover the culture dish with a lid, and culture in a constant temperature dark environment to obtain the primary screening colonies; The components of the solid LB medium in step 2-3 are 10 g / L tryptone, 5 g / L yeast extract, 15 g / L sodium chloride, and 18 g / L agar.

4. A bacterial agent, characterized in that The bacterial agent is the Bacillus Velezii of claim 1, or includes the Bacillus Velezii of claim 1.

5. A fermentation agent, characterized in that: The fermentation agent is prepared by fermentation of Bacillus Velezii according to claim 1, and the fermentation steps are: Step 1, prepare fermentation medium: corn flour 20-22.5g / L, glucose 2.5-5g / L, soybean meal 5-7.5g / L, magnesium sulfate 0.25-0.5g / L, potassium dihydrogen phosphate 1-2g / L, potassium monohydrogen phosphate 0.5-1g / L; Step 2, inoculating the above-mentioned Bacillus Velez subtilis into the fermentation medium in step 1, with the inoculation amount being 1-10% of the total volume of the fermentation medium, and fermenting at 28-30° C. for 48 hours; Step 3: Filter the fermented bacterial liquid to obtain the fermentation agent.

6. A microbial agent for inhibiting pathogens, characterized in that: The microbial agent includes one or more of the Bacillus Velezii of claim 1, the agent of claim 4, or the fermentation agent of claim 5.

7. A growth-promoting agent, characterized in that: The growth promoter includes one or more of the Bacillus Velezii of claim 1, the bacterial agent of claim 4, or the fermentation bacterial agent of claim 5.

8. A yield-increasing agent, characterized in that: The yield-increasing agent comprises one or more of the Bacillus Velezii of claim 1, the bacterial agent of claim 4, or the fermentation bacterial agent of claim 5.

9. A soil conditioner, characterized in that: The soil conditioner comprises one or more of the Bacillus Velezii of claim 1, the bacterial agent of claim 4, or the fermentation bacterial agent of claim 5.

10. A biological organic fertilizer, characterized in that: The biological organic fertilizer comprises one or more of the Bacillus Velezii of claim 1, the bacterial agent of claim 4, or the fermentation bacterial agent of claim 5; The biological organic fertilizer also includes biochar and Cnidium monnieri.

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