Bacillus velezensis and application thereof
By using bacterial suspensions and fermentation broths prepared from Bacillus vesiculosus, the problems of preventing and controlling fungal diseases in various crops and promoting crop rooting and germination have been solved, achieving the effects of disease control and crop growth promotion in crops such as grapes, apples, and tobacco.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- APAXFON (BAOTOU) BIOLOGICAL SCI & TECH CO LTD
- Filing Date
- 2022-09-05
- Publication Date
- 2026-08-04
AI Technical Summary
Existing technologies are insufficient to effectively control various fungal diseases in crops such as grapes, apples, and tobacco, and there is a lack of effective methods to promote crop rooting and germination.
This invention provides Bacillus belye and its prepared bacterial suspension and sterile fermentation broth for the preparation of microbial agents and microbial fertilizers, which can be used to prevent and control various fungal diseases and promote the germination of wheat, corn, tomatoes and peppers.
Bacillus berleis significantly inhibits fungal diseases in crops such as grapes, apples, and tobacco, enhances crop resistance, and promotes root growth and germination.
Smart Images
Figure CN115851493B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, and in particular relates to a Bacillus belye and its applications. Background Technology
[0002] Bacillus velezensis, two strains, CR.14b and CR.502T, were isolated from soil samples taken from the mouth of the Velez River in Mahraga Province, southern Spain, in 1999–2000. This bacterium belongs to the phylum Firmicutes, order Bacillusales, family Bacillusaceae, and genus Bacillus. It is a type of spore-forming Gram-positive bacterium. Most Bacillus species possess advantages such as broad antibacterial spectrum, rapid growth, ease of isolation and culture, strong stress resistance, and high biocompatibility, making them widely studied as probiotics in agriculture, food, industry, medicine, metallurgy, forestry, environmental protection, and military fields. Summary of the Invention
[0003] This invention provides a Bacillus velezensis, which was deposited on May 30, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; the accession number is CGMCC NO.24988; and the taxonomic name is Bacillus velezensis.
[0004] Another aspect of the present invention provides a bacterial suspension prepared from the above-mentioned Bacillus berleis.
[0005] Another aspect of the present invention provides a sterile fermentation broth prepared from the above-mentioned Bacillus beryl and / or a bacterial suspension prepared using the above-mentioned Bacillus beryl.
[0006] Another aspect of the present invention provides a microbial agent, which includes the above-mentioned Bacillus berleis or a bacterial suspension prepared from the above-mentioned Bacillus berleis or the above-mentioned sterile fermentation broth.
[0007] Preferably, the microbial agent is a solid agent or a liquid agent.
[0008] Another aspect of the present invention provides a microbial fertilizer, which includes the above-mentioned Bacillus berberis, the above-mentioned bacterial suspension, the above-mentioned sterile fermentation broth, or the above-mentioned microbial agent.
[0009] Another aspect of the present invention provides the application of the above-mentioned Bacillus belyes or the bacterial suspension prepared from the above-mentioned Bacillus belyes in the control of Botrytis cinerea, Coniella diplodiella, Colletotrichum gloeosporioides, Lasiodiplodiatheobromae, Alternaria alternata apple pathotype, Botryosphaeria dothidea, Colletotrichum acutatum, Monilinia fructicola, or Phytophthora nicotianae.
[0010] Another aspect of the present invention provides the application of the above-mentioned sterile fermentation broth in the prevention and control of grape gray mold (Botrytiscinerea), grape white rot (Coniella diplodiella), grape anthracnose (Colletotrichum gloeosporioides), grape canker (Lasiodiplodia theobromae), apple leaf spot (Alternaria alternata apple pathotype), apple ring rot (Botryosphaeria dothidea), apple anthracnose (Colletotrichum acutatum), cherry brown rot (Monilinia fructicola), or tobacco Phytophthora nicotianae.
[0011] Another aspect of the present invention provides the application of the above-mentioned Bacillus belye or the bacterial suspension prepared from the above-mentioned Bacillus belye in promoting the germination of wheat, corn, tomato, and pepper.
[0012] Another aspect of the present invention provides the application of the above-mentioned sterile fermentation broth in promoting the germination of wheat, corn, tomatoes, and peppers.
[0013] Compared with the prior art, the present invention has the following beneficial effects:
[0014] The Bacillus berberis provided by this invention has a good inhibitory effect on diseases of grapes and potatoes, and has a good root and shoot promotion effect during the seedling stage. When used as a special fertilizer, this strain can significantly improve the crop's stress resistance and help the crop to take root during the seedling stage.
[0015] Biological Preservation Instructions: Preservation of microbial strains used for patent applications.
[0016] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;
[0017] Accession number: CGMCC NO.24988;
[0018] Deposit date: May 30, 2022;
[0019] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;
[0020] Taxonomic name: Bacillus velezensis. Attached Figure Description
[0021] Figure 1 This is a diagram showing the antibacterial effect of Bacillus belyssus in Example 1.
[0022] Figure 2 This is the phylogenetic tree of pathogen YV01 in Example 2. Detailed Implementation
[0023] Example 1
[0024] This embodiment provides the screening process for Bacillus belyssus:
[0025] A. Materials and Reagents
[0026] Fertilizer fermentation stock solution; Pathogens: *Botrytis cinerea* (grape gray mold), *Coniella diplodiella* (grape white rot), *Colletotrichum gloeosporioides* (grape anthracnose), *Lasiodiplodia theobromae* (grape canker), *Alternaria alternata* (apple leaf spot), *Botryosphaeria dothidea* (apple ring rot), *Colletotrichumacutatum* (apple anthracnose), *Monilinia fructicola* (cherry brown rot), *Phytophthoranicotianae* (tobacco blight). NA medium, PDA medium.
[0027] B. Test Methods
[0028] NA medium (g / L): 33g of NA medium powder, add distilled water to 1L, pH 7.2-7.4.
[0029] PDA medium (g / L): 46g of PDA medium powder, add distilled water to 1L, pH 6.5~7.5.
[0030] Nine pathogenic bacteria were activated and inoculated onto PDA medium. After incubation at 25°C for 5 days, samples were collected from the edge of the colonies. The mushroom cakes are placed in a refrigerator at 4°C for later use.
[0031] C. Isolation of antagonistic microorganisms
[0032] 200 μL of BGF fermentation broth was evenly spread on a PDA plate. A bacterial cake was picked and inoculated in the center of the plate. After culturing at 25°C for 5 days, a colony that inhibited the growth of pathogenic bacteria was observed. The colony was isolated and purified, numbered YV01, and then stored in a refrigerator at 4°C.
[0033] D. Antagonistic microbial confrontation experiment
[0034] Select a bacterial disc of pathogen YV01 and inoculate it in the center of an agar plate. Using the pathogen as the center, draw a cross on the back of the plate with a radius of 2.5 cm. Use an inoculation needle to spot-inoculate the purified bacterial strain at four points on each of the four crosses. Incubate the PDA plates at 25°C for 3–7 days. Determine the antagonistic effect of the strain by observing the appearance of an antagonistic zone (inhibition zone) on the plate, and record the diameter of the inhibition zone. Each treatment was repeated in triplicate. The inhibition rate after 7 days of incubation is shown in Table 1, and the confrontation results are shown in [Table 2]. Figure 1 .
[0035]
[0036] Table 1. Inhibition rate of Bacillus belye against 9 pathogenic fungi
[0037] Apple ring rot pathogen 53.0％ Grape ulcer pathogen 67.6％ Grape gray mold pathogen 64.7％ Grape white rot pathogen 71.5％ Apple spot pathogen 56.7％ Phytophthora indicum 50.6％ Apple anthracnose pathogen 54.6％ Cherry brown rot pathogen 59.5％ Grape anthracnose pathogen 58.7％
[0038] Example 2
[0039] The morphology of the isolated strain was observed by Gram staining, and physiological and biochemical tests were conducted according to the "Handbook of Systematic Identification of Common Bacteria". These tests showed that the isolated strain could decompose and utilize glucose, lactose, sucrose, fructose, mannitol, and maltose; it could hydrolyze starch and gelatin; and it could utilize glucose to produce acid. Its oxidase reaction, VP reaction, nitrate reduction reaction, and catalase reaction were all positive, while the methyl red test and H2S reaction were negative. Based on the culture characteristics, morphology, and physiological and biochemical features of this strain, it was preliminarily identified as a member of the genus *Bacillus*.
[0040] 16S rDNA sequencing and assembly results of the bacterial strain:
[0041] SEQ ID NO.1:
[0042]
[0043] Construct a phylogenetic tree of pathogen YV01, see [link / reference]. Figure 2 The pathogen YV01 was identified as Bacillus velezensis.
[0044] Example 3
[0045] This embodiment tested the growth-promoting effect of Bacillus belyssus in Example 2. The specific process is as follows:
[0046] (1) Liquid fertilizer: produced by Yuantaifeng (Baotou) Biotechnology Co., Ltd., trade name: Mineral Source Black Gold Life Liquid. Diluted 100 times and used for germination test.
[0047] (2) LB medium: 1% tryptone, 0.5% yeast extract, 1% sodium chloride, diluted with distilled water to 1L, pH 7.2–7.4; Liquid fertilizer LB medium: 1% tryptone, 0.5% yeast extract, 1% sodium chloride, diluted with liquid fertilizer to 1L, pH 7.2–7.4. NA medium: 1% peptone, 0.3% beef extract, 0.5% sodium chloride, 1.6% agar, diluted with distilled water to 1L, pH 7–7.5.
[0048] (3) Fermentation of Bacillus belye seed culture: Using a toothpick or a 100μL pipette tip, take a small amount of Bacillus belye culture stored on NA medium at 4℃ and place it in an Erlenmeyer flask containing 20ml LB medium. Ferment at 37℃ and 200rpm. Examine the seed culture under a microscope. When it reaches the logarithmic growth phase, inoculate the seed culture into the fermentation flask.
[0049] (4) Take the seed liquid of Bacillus belysinus and inoculate it into 500 mL Erlenmeyer flasks containing 50 mL LB medium and 50 mL liquid fertilizer LB medium respectively at a ratio of 10%. After fermentation at 200 rpm and 37℃ for 48 h, Bacillus belysinus fermentation broth (water) and Bacillus belysinus fermentation broth (liquid fertilizer) are obtained respectively.
[0050] (5) Examine the fermentation broth under a microscope after 48 hours to observe whether spores have been produced. Stop fermentation when 90% of spores have been produced. Centrifuge the fermentation broth at 8000 rpm for 10 minutes and collect the supernatant.
[0051] (6) Dilute the supernatant 100 times and use it for germination test.
[0052] (7) Three parallel tests were performed for each sample. Two qualitative filter papers were placed in each petri dish, 10 ml of supernatant diluent was added, and 20 wheat seeds were placed in the dish and cultured at 25°C in the room. At the same time, wheat seeds were cultured with pure water as a control blank test.
[0053]
[0054]
[0055] Table 2. Germination test results of Bacillus belyss fermentation broth.
[0056]
[0057] Example 4
[0058] 1. Test materials
[0059] Bacillus belye seed culture: Using a toothpick or a 100μL pipette tip, take a small amount of Bacillus belye culture stored on NA medium at 4℃ and place it in an Erlenmeyer flask containing 20ml LB medium. Ferment at 37℃ and 200rpm. Examine the seed culture under a microscope. When it reaches the logarithmic growth phase, inoculate the seed culture into a fermentation flask.
[0060] Bacillus belysin fermentation broth: Take the Bacillus belysin seed liquid and inoculate it into 500 mL Erlenmeyer flasks containing 50 mL LB medium at a ratio of 10%. Ferment at 200 rpm and 37 °C for 48 h to obtain Bacillus belysin fermentation broth.
[0061] After 48 hours, the fermentation broth was examined under a microscope to observe whether spores had formed. Fermentation was stopped when 90% of the spores had formed. The fermentation broth was centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected.
[0062] 2. Experimental Procedure
[0063] Several seeds of corn (Green Superman), tomato (Diana), and pepper (Resistant to Continuous Cropping Chili) were taken respectively. The seeds were soaked in the supernatant of Bacillus vesiculosus fermentation broth for 30 minutes. CK used water instead of the sterile fermentation broth. The soaked seeds were then planted in flowerpots. When the corn reached the "two leaves and one bud" stage, and the other seeds reached the corresponding stage, and the seedlings of the same species showed similar growth, a suspension of gray mold spores was sprayed onto the plant leaves using a small spray bottle. The disease incidence and severity were observed and recorded after 10 days.
[0064] Leaf disease severity levels: Level 0: No leaves are diseased; Level 1: Diseased leaves account for 5% of the total number of leaves; Level 3: Diseased leaves account for 10% of the total number of leaves; Level 5: Diseased leaves account for 30% of the total number of leaves; Level 7: Diseased leaves account for 50% of the total number of leaves; Level 3: Diseased leaves account for more than 50% of the total number of leaves.
[0065] 3. Test Results
[0066] Table 3 Seedling Stage of Different Crops
[0067]
[0068] As shown in Table 3, seeds soaked in Bacillus venetum fermentation broth exhibited more developed root systems during the seedling stage, resulting in greater crop biomass. Furthermore, the incidence of disease was less severe after spraying with pathogen spore liquid compared to treatment with plain water.
[0069] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A Bacillus velezensis, characterized in that, The Bacillus velezensis described was deposited on May 30, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; accession number: CGMCC NO.24988; taxonomic name: Bacillus velezensis.
2. A bacterial suspension prepared using Bacillus belye as described in claim 1.
3. A microbial inoculant, characterized in that, The microbial agent includes Bacillus berleis as described in claim 1 or the bacterial suspension as described in claim 2.
4. The microbial inoculant of claim 3, wherein, The microbial agent can be a solid agent or a liquid agent.
5. A microbial inoculant, characterized in that, The microbial fertilizer includes Bacillus berberis as described in claim 1, the bacterial suspension as described in claim 2, or the microbial agent as described in claim 3.
6. The application of the *Bacillus belyssus* of claim 1 or the bacterial suspension prepared from *Bacillus belyssus* of claim 2 in the prevention and control of *Botrytis cinerea*, *Coniella diplodiella*, *Colletotrichum gloeosporioides*, *Lasiodiplodiatheobromae*, *Alternaria alternata*, *Botryosphaeria dothidea*, *Colletotrichum acutatum*, *Monilinia fructicola*, or *Phytophthora nicotianae*.
7. The application of the bacterial suspension prepared from Bacillus vesiculosus of claim 1 or Bacillus vesiculosus of claim 2 in promoting the germination of wheat, corn, tomato, and pepper.