Bacillus velezensis Ld2112 and application thereof

Abstract

The invention provides a bacillus velezensis Ld2112 strain and application thereof. The bacillus velezensis Ld2112 strain is firstly separated from rhizosphere soil of grapes, and the preservation number of the strain is CCTCC (China Center for Type Culture Collection) NO: M 2025831. The strain fermentation liquor has a good prevention effect on grape white rot, and the prevention effect reaches 75% or above; the composition has a good prevention effect and a good treatment effect on anthracnose caused by colletotrichum gloeosporioides, and the prevention effect and the treatment effect are 89.47% and 84.66% respectively; the compound has a good treatment effect on anthracnose caused by colletotrichum gloeosporioides, the treatment effect reaches 70% or above, and the compound can be used as a biopesticide for preventing and treating grape white rot and / or grape anthracnose.

Description

Technical Field

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

[0002] Bacillus velez ( Bacillus velezensis ) is a new species of the genus Bacillus. Its metabolites possess broad-spectrum antimicrobial activity, making it an important biocontrol agent in plant protection and forestry. Bacillus velezensis has complex and diverse mechanisms of action against plant diseases, including antagonism, lysis, induction of plant resistance, competition, and promotion of plant growth. Bacillus velezensis can produce a variety of secondary metabolites, including lipopeptides, antimicrobial proteins, polyketides, volatile substances, and bacteriocins, which directly or indirectly inhibit pathogen growth. It can also produce lytic substances that destroy the pathogen's cell wall and disintegrate the mycelium. Since its discovery in 2016, Bacillus velezensis has become a research hotspot in recent years.

[0003] Microbial biopesticides have become a research hotspot in the biopesticide field and a primary choice for biopesticide registration due to their rapid reproduction, long-lasting control effects, diverse development and utilization pathways, resistance to resistance, simple fermentation processes, and low production costs. Bacillus sp. can form endospores, survive heat exposure and desiccation, and can be formulated into stable dry powders with long shelf lives, making them an ideal candidate for microbial pesticide formulation development. Currently, five strains of Bacillus velezensis have been registered as pesticides: CGMCC No. 14384, C17271, M173, LW-6, and 9912. Formulations include suspension concentrates, wettable powders, and water-dispersible granules, as well as both parent and formulations. Currently registered Bacillus velezensis strains are primarily used to control tobacco powdery mildew, brown spot, and black shank; cucumber powdery mildew; citrus canker; rice bacterial leaf streak; tomato gray mold; cucumber bacterial angular leaf spot; Panax notoginseng, ginseng, and strawberry root rot; and apple rot. At present, there are no reports on the use of Bacillus Velezii to control grape white rot and anthracnose.

[0004] Grape white rot and anthracnose are the main diseases that harm grape production. Grape white rot is the most important disease that causes fruit rot during the grape growing period. It is caused by Aspergillus niger ( Coniella diplodiella ) infection, which mainly harms the grape bunches. After the disease occurs, the infected fruit gradually shrinks, the berries or bunches are easy to fall off, and the grape yield is seriously reduced. Grape anthracnose is the main disease of grapes, which is caused by the cryptic anthracnose fungus ( C. aenigma ), Colletotrichum gloeosporioides ( C. gloeosporioidesWhite rot and anthracnose are common in grapes and are the main diseases of the grapevine. The disease begins in the early stages of flowering, causing infected flowers and pedicels to turn brown and rot, leading to massive flower drop and, later, the entire flower cluster to rot. Therefore, the prevention and control of grapevine white rot and anthracnose is of great importance. Summary of the Invention

[0005] Based on this, the present invention provides a strain of Bacillus velez Ld2112 and its application. The present invention first isolates and obtains Bacillus velez Ld2112 from grape rhizosphere soil. Bacillus velezensis ) Ld2112, this strain can be used as a biological pesticide to control grape white rot and / or grape anthracnose, and has good application prospects.

[0006] The above-mentioned object of the present invention is achieved through the following technical solutions: the Bacillus velezensis provided by the present invention ( Bacillus velezensis Strain Ld2112 was isolated from the rhizosphere soil of a grapevine in a vineyard in Yantai City, Shandong Province. It was deposited with the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China, on April 21, 2025, under the accession number CCTCC NO: M 2025831. It exhibits the following microbiological characteristics: On NA medium, Ld2112 colonies are milky white, irregular in shape, opaque, with irregular margins, a central white protrusion, and a matte surface. NA medium is recommended for standard culture, and 20% glycerol is recommended for long-term storage.

[0007] The present invention first provides a fermentation method for Bacillus velezensis Ld2112, comprising preparing a seed solution and fermenting the solution to obtain a fermentation solution. The fermentation solution is cultured under the following conditions: an inoculum size of 3-10% (by weight), a culture temperature of 30-35°C, and a culture time of 24-48 hours. The fermentation medium comprises (by weight): 3.5% soybean meal, 2.1% corn starch, 0.5% glucose, 0.02% manganese sulfate, and the remainder water, with a pH of 7.0-7.5.

[0008] The present invention also provides a microbial agent with Bacillus velezensis Ld2112 as an active ingredient. The microbial agent can be a fermentation broth obtained by the above method, or a bacterial powder prepared by further adding a carrier, or various dosage forms prepared by adding different adjuvants, such as a suspension, a wettable powder, a water-dispersible granule, etc.

[0009] The present invention also provides the use of the Bacillus velezensis Ld2112 strain or its microbial agent (fermentation broth is used in the embodiment) for preventing and treating grape white rot and / or grape anthracnose.

[0010] The above-mentioned grape anthracnose includes grape anthracnose caused by Colletotrichum gloeosporioides and / or Colletotrichum crypticum.

[0011] The present invention also provides a method for preventing and controlling grape white rot and / or grape anthracnose, comprising applying the above-mentioned microbial agent (fermentation broth is used in the embodiment) to grapes with white rot and / or anthracnose.

[0012] The present invention demonstrates the following technical advantages: Using a spray treatment method, the fermentation broth of the Bacillus Velez Ld2112 strain demonstrated a superior preventive effect against grape white rot, exceeding 75%, compared to pure water and a control agent. It also demonstrated superior preventive and therapeutic effects against anthracnose caused by Colletotrichum crypticum, reaching 89.47% and 84.66%, respectively. Furthermore, it demonstrated a superior therapeutic effect against anthracnose caused by Colletotrichum gloeosporioides, reaching over 70%. Because the Bacillus Velez Ld2112 agent produces no residual residue and has no toxic side effects, it can reduce the use of chemical pesticides. Overall, the Bacillus Velez Ld2112 strain has significant potential for application in the biological control of grape white rot and / or grape anthracnose. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the single colony morphology of Bacillus velezinoff Ld2112 strain; Figure 2 Figures 1 and 2 show the indoor antibacterial effect of Bacillus velezensis Ld2112 strain; Figure A shows the antibacterial effect against Aspergillus niger; Figure B shows the antibacterial effect against Colletotrichum occidentalis; and Figure C shows the antibacterial effect against Colletotrichum gloeosporioides. The upper panels of Figures A, B, and C represent the treatment group, and the lower panels represent the control group. Figure 3 Figures show the preventive and therapeutic effects of Bacillus Velez Ld2112 fermentation broth on grape white rot. Panel A shows the group treated with Bacillus Velez Ld2112 fermentation broth, Panel B shows the control treatment group, and Panel C shows the water treatment group. The upper panels of Panels A, B, and C show the preventive treatment, while the lower panels show the therapeutic treatment. Figure 4 Figures show the preventive and therapeutic effects of Bacillus Velez Ld2112 fermentation broth on grape anthracnose caused by Colletotrichum gloeosporioides. Panel A shows the group treated with Bacillus Velez Ld2112 fermentation broth, Panel B shows the control treatment group, and Panel C shows the water treatment group. The upper panels of Panels A, B, and C show the preventive treatment, while the lower panels show the therapeutic treatment. Figure 5 These are diagrams showing the preventive and therapeutic effects of the fermentation broth of Bacillus Velez Ld2112 on grape anthracnose caused by Colletotrichum crypticum. Figure A shows the group treated with the fermentation broth of Bacillus Velez Ld2112, Figure B shows the group treated with the control agent, and Figure C shows the group treated with clean water. The upper figures of Figures A, B, and C show the preventive treatment, and the lower figures show the therapeutic treatment. DETAILED DESCRIPTION

[0014] The present invention will be further described below with reference to specific embodiments and the accompanying drawings. The advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely illustrative and do not constitute any limitation on the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0015] Example 1: Isolation and identification of Bacillus velezinis Ld2112 strain 1. Strain Isolation: The Bacillus Velez is isolated from rhizosphere soil of grapes in a vineyard in Yantai City, Shandong Province by using a soil dilution separation method. The isolation method is as follows: (1) Soil sample collection: After cleaning the topsoil, sample 200 g of soil from 0-20 cm around the rhizosphere, perform a preliminary sieve (to remove impurities in the soil, such as stones, plant roots, etc.), and take 10 g of soil sample for separation; (2) Weigh 10 g of soil sample, add 90 mL of sterile water, place in a shaker at 28°C and 180 r / min for 30 min, take out and let it stand in a conical flask for about 30 min, and dilute it into 10 -1 -10 -9 g / mL soil suspension; (3) Take the concentration of 10 -6 , 10 -7 , 10 -8 , 10 -9 100 μL of the soil suspension at 100 μg / mL was dropped onto a PDA medium plate, spread with a glass rod, and cultured at a constant temperature of 28-30°C for 2-3 days; (4) Use an inoculation needle to pick up single colonies with different morphologies, purify them by plate streak method, and culture them in a 28-30°C incubator. At the same time, the isolated different strains are numbered and transferred to slant culture medium for later use; the culture medium used for purification is PDA medium; (5) Using grape white rot pathogen as the target, a strain with strong inhibitory activity against grape white rot pathogen was screened by plate confrontation method and numbered Ld2112.

[0016] The PDA culture medium formula is: 200 g potatoes (peeled and cut into pieces), 20 g glucose, 14 g agar, and 1000 mL distilled water.

[0017] 2. Strain Identification (1) Microbiological characteristics: On NA medium, the colonies of strain Ld2112 are milky white, irregular in shape, opaque, with irregular edges, white protrusions in the middle, and a matte surface (see Figure 1 ).

[0018] (2) Molecular biological characteristics: The 16S rRNA gene sequence of the strain Ld2112 of the present invention is shown in SEQ No. 1; gyrA The gene sequence determination result is shown in SEQ No.2.

[0019] The 16S rRNA amplified sequence of strain Ld2112 and a housekeeping gene ( gyrA ) sequences were compared in the NCBI database, and the 16S rRNA sequences were compared with Bacillus velezensis FWJ16X (OK147625) has a homology of 99.93%, gyrA Base and Bacillus velezensis The homology of Hx05 (CP029473) was 99.88%. Combined with the morphological characteristics of Ld2112 strain, it was determined that Ld2112 belonged to Bacillus velezensis ( Bacillus velezensis ).

[0020] This strain was deposited in the China Center for Type Culture Collection (CCTCC) on April 21, 2025, at Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M 2025831.

[0021] Example 2: Indoor verification of the effect of Bacillus velezensis Ld2112 against grape white rot and anthracnose 1. Experimental Methods The plate confrontation culture method was used to culture the grape white rot pathogen Aspergillus niger ( Coniella diplodiella ), grape anthracnose, including Colletotrichum crypticum ( C. aenigma ), Colletotrichum gloeosporioides ( C. gloeosporioides ss) bacterial cakes (9 mm) were transferred to the center of each plate. Bacillus velezensis Ld2112 was inoculated symmetrically 2 cm from the cakes and incubated in a 28°C biochemical incubator. Each treatment was replicated three times. When mycelium in the control group (inoculated only with the 9 mm bacterial cake in the center of the plate) filled the plate, the colony diameter was measured using the cross-hatch method, and the relative inhibition rate was calculated.

[0022] 2. Experimental Results From Table 1 and Figure 2As can be seen, the inhibition rates of Bacillus Velez Ld2112 on the mycelial growth of Aspergillus niger, Colletotrichum crypticum, and Colletotrichum gloeosporioides were 82.26%, 77.26%, and 78.45%, respectively. Therefore, Bacillus Velez Ld2112 has a strong inhibitory effect on the mycelial growth of grape white rot pathogens and anthracnose pathogens.

[0023] Table 1 Indoor antibacterial activity test of Bacillus velezensis Ld2112 against Colletotrichum gloeosporioides, Colletotrichum crypticum and Colletotrichum gloeosporioides

[0024] Example 3: Preparation of fermentation broth of Bacillus velezinoff Ld2112 Seed liquid culture medium: 3 g beef extract, 5 g peptone, 5 g sodium chloride, 1000 mL water, pH 7.3.

[0025] Fermentation medium (weight percentage): soybean meal 3.5%, corn starch 2.1%, glucose 0.5%, manganese sulfate 0.02%, the rest is water, pH 7.0-7.5.

[0026] Fermentation method: 1) Shaking culture: Pick a small number of Bacillus velezensis Ld2112 colonies from the slant of the test tube and transfer them to the seed liquid culture medium. Incubate the culture in a shaking incubator at 30°C for 12 hours to obtain the seed liquid. 2) Fermentation culture: Add the seed liquid cultured on a shaking table to the fermentation medium for culture at a temperature of 32°C, an initial pH of 7.0-7.5, a liquid filling volume of 70%, an inoculation volume of 10%, a rotation speed of 200 r / min, and a culture time of 36 hours. The bacterial count can reach 10-30 billion / g.

[0027] Example 4: Verification of the effect of fermentation broth of Bacillus velez Ld2112 on grape white rot This example provides experiments related to the treatment of grape white rot with the fermentation broth of Bacillus velez Ld2112.

[0028] 1. Experimental Methods 1.1 Test agent: Fermentation broth of Bacillus velezensis Ld2112 (active cells 20 billion / g).

[0029] 1.2 Test crops and control targets: The test crop was Kyoho grapes; the target was white rot. Unexposed grape berries of consistent size and health were collected from the experimental base. They were rinsed three times with sterile water, air-dried, and placed in a fresh-keeping box (16 cm × 10 cm × 6 cm) containing soaked filter paper until ready for use.

[0030] 1.3 Methods 1.3.1 Determination of preventive effect The fermentation liquid of Bacillus velezensis Ld2112 was sprayed onto the surface of grape berries using a hand sprayer (E532) to form a uniform film. Grape berries sprayed with water served as the control group, while 40% prochloraz emulsion (1000-fold dilution) served as the control agent group. 24 h after treatment, the surface of the grape berries was slightly punctured with a sterilized inoculation needle to form a microwound. 100 μL of the spore suspension of Aspergillus niger (10 6 Spores / mL) were dropped onto the wound. Ten grape berries were treated in each treatment, and one inoculation site was established on each berry. The inoculated berries were placed in an incubator (25°C; L﹕D = 12 h﹕12 h; relative humidity above 85%) for incubation and observation of the disease. The disease index and control efficacy were calculated after 7 days of incubation in the incubator. The experiment was repeated four times.

[0031] 1.3.2 Determination of treatment efficacy Grape berries collected for the experiment were first inoculated with a spore suspension of Aspergillus niger according to the above method. Twenty-four hours later, the same concentration of the suspension was sprayed as described above. Control berries were sprayed with water and treated with a 40% prochloraz emulsion (1000-fold dilution) as a control. Ten berries were inoculated at one site per treatment. The experimental grapes were incubated in an incubator (25°C; L / D = 12 h:12 h; relative humidity above 85%) and observed for disease development. After seven days of incubation, the disease index and control efficacy were calculated. The experiment was repeated four times.

[0032] 1.4 Statistics The grading standards for grape white rot are shown in Table 2.

[0033] Table 2 Grape white rot classification standards

[0034] Disease index = 100 × ∑ [number of diseased leaves (fruits) at each level × relative level value] / [total number of leaves (fruits) surveyed × highest level value]; Control effect (%) = (control disease index - treatment disease index) / control disease index × 100.

[0035] 2. Experimental Results Table 3 and Figure 3 Results showed that the fermentation broth of Bacillus velezensis Ld2112 had a significant preventive effect against grape white rot caused by Aspergillus albicans, achieving a 75.93% efficacy. The control agent, a 1000-fold dilution of 40% prochloraz, had a preventive effect of 82.41%. The fermentation broth of Bacillus velezensis Ld2112 and the control agent had moderate therapeutic effects, achieving only 43.52% and 63.89% efficacy, respectively.

[0036] Table 3 Control effect of fermentation broth of Bacillus velezensis Ld2112 on grape white rot caused by Aspergillus albicans

[0037] 3. Summary The fermentation broth of Bacillus velezensis Ld2112 had a good preventive effect on grape white rot caused by Aspergillus albicans, reaching 75.93%, with no significant difference compared with the control agent (P<0.05).

[0038] Example 5: Verification of the effect of fermentation broth of Bacillus velezensis Ld2112 on grape anthracnose caused by Colletotrichum gloeosporioides This example provides experiments related to the use of fermentation broth of Bacillus velezensis Ld2112 against grape anthracnose caused by Colletotrichum gloeosporioides.

[0039] 1. Experimental Methods 1.1 Test agent: Fermentation broth of Bacillus velezensis Ld2112 (active cells 20 billion / g).

[0040] 1.2 Test crops and control targets: The test crop was Summer Black grapes; the target disease was anthracnose caused by Colletotrichum gloeosporioides. Uninfected grape berries of uniform size and health were collected from the experimental base. They were rinsed three times with sterile water, air-dried, and placed in a fresh-keeping box (16 cm × 10 cm × 6 cm) containing soaked filter paper until ready for use.

[0041] 1.3 Methods 1.3.1 Determination of preventive effect The fermentation liquid of Bacillus velezensis Ld2112 was sprayed onto the surface of grape berries using a hand sprayer (E532) to form a uniform film. Grape berries sprayed with water served as the control group, while those treated with 40% prochloraz emulsion (1000-fold dilution) served as the control agent group. 24 h after treatment, the surface of the grape berries was slightly punctured with a sterilized inoculation needle to form a microwound. 100 μL of spore suspension of Colletotrichum gloeosporioides (10 6 Spores / mL) were dropped onto the wound. Ten grape berries were treated in each treatment, and one inoculation site was established on each berry. The inoculated berries were placed in an incubator (25°C; L﹕D = 12 h﹕12 h; relative humidity above 85%) for incubation and observation of the disease. The disease index and control efficacy were calculated after 7 days of incubation in the incubator. The experiment was repeated four times.

[0042] 1.3.2 Determination of treatment efficacy Grape berries were inoculated with a spore suspension of Colletotrichum gloeosporioides according to the above method. 24 hours later, the same concentration of the drug solution was sprayed as described above. Control berries were sprayed with water and treated with a 40% prochloraz emulsion (1000-fold dilution) as a control. Ten berries were inoculated at one site per treatment. The experimental grapes were incubated in an incubator (25°C; L / D = 12h:12h; relative humidity above 85%) and observed for disease development. After seven days of incubation, the disease index and control efficacy were calculated. The experiment was repeated four times.

[0043] 1.4 Statistics Grape anthracnose is graded based on the extent of the diseased area. The grading criteria are shown in Table 4.

[0044] Table 4 Grading standards for grape anthracnose

[0045] The disease index and the calculation formula for the prevention and treatment effect are the same as in Example 4.

[0046] 2. Experimental Results From Table 5 and Figure 4 It can be seen that the preventive and therapeutic effects of the fermentation broth of Bacillus Velezii Ld2112 strain on grape anthracnose caused by Colletotrichum gloeosporioides are 66.67% and 71.11%, respectively. The preventive and therapeutic effects of the control agent 1000 times diluted 40% prochloraz in water emulsion are 75.56% and 76.30%, respectively.

[0047] Table 5 Control effect of fermentation broth of Bacillus velez Ld2112 on grape anthracnose caused by Colletotrichum gloeosporioides

[0048] 3. Summary The fermentation broth of Bacillus Velez strain Ld2112 has a good preventive and therapeutic effect on grape anthracnose caused by Colletotrichum gloeosporioides, which is equivalent to the control agent and can effectively treat the damage caused by Colletotrichum gloeosporioides.

[0049] Example 6: Verification of the effect of fermentation broth of Bacillus velez Ld2112 on grape anthracnose caused by Colletotrichum crypticum This example provides experiments related to the use of fermentation broth of Bacillus velez Ld2112 against grape anthracnose caused by Colletotrichum crypticum.

[0050] 1. Experimental Methods Target of prevention and control: Anthrax caused by Colletotrichum crypticum; During the experiment, Colletotrichum crypticum spore suspension (10 6 spores / mL) instead of the Colletotrichum gloeosporioides spore suspension, and the rest was the same as in Example 5.

[0051] 2. Experimental Results Table 6 and Figure 5 The results showed that the fermentation broth of Bacillus Velez Ld2112 had a significant preventive and therapeutic effect against grape anthracnose caused by Colletotrichum crypticum, achieving efficacy rates of 89.47% and 84.66%, respectively. The control agent achieved efficacy rates of 79.73% and 85.78%, respectively. In terms of therapeutic efficacy, the fermentation broth of Bacillus Velez Ld2112 and the control agent were essentially equivalent. Furthermore, the fermentation broth of Bacillus Velez Ld2112 was significantly more effective than the control agent in preventing Colletotrichum crypticum from infecting grapes.

[0052] Table 6 Control effect of fermentation broth of Bacillus velez Ld2112 on grape anthracnose caused by Colletotrichum crypticum

[0053] 3. Summary The fermentation broth of Bacillus Velez Ld2112 has a good preventive and therapeutic effect against grape anthracnose caused by Colletotrichum crypticum. It is significantly more effective than the control agent in preventing grape anthracnose damage; in terms of treatment, the fermentation broth of Ld2112 is equivalent to the control agent. Bacillus Velez Ld2112 has good application prospects in the prevention and treatment of grape anthracnose caused by Colletotrichum crypticum.

Claims

1. A strain of Bacillus velezinoffii ( Bacillus velezensis ) Ld2112, the deposit number of the strain is CCTCC NO: M 2025831.

2. The fermentation method of the Bacillus velezensis Ld2112 strain according to claim 1, comprising preparing a seed solution and fermenting and culturing the fermentation solution, wherein: The culture conditions of the fermentation culture are: inoculation amount 3-10%, culture temperature 30-35° C., and culture time 24-48 hours.

3. The fermentation method according to claim 2, wherein: The fermentation medium comprises, by weight percentage, 3.5% soybean meal powder, 2.1% corn starch, 0.5% glucose, 0.02% manganese sulfate, and the remainder water, with a pH of 7.0-7.

5.

4. A microbial agent with the Bacillus Velezii Ld2112 according to claim 1 as an active ingredient, characterized in that: The microbial agent is prepared from the fermentation liquid obtained by the fermentation method according to claim 2 or 3.

5. Use of the Bacillus Velez strain Ld2112 according to claim 1 or the microbial agent according to claim 4 for preventing and treating grape white rot and / or grape anthracnose.

6. The use according to claim 5, characterized in that: The grape anthracnose includes grape anthracnose caused by Colletotrichum gloeosporioides and / or Colletotrichum crypticum.

7. A method for preventing and controlling grape white rot and / or grape anthracnose, characterized in that: The method comprises applying the microbial agent according to claim 4 to grapes having grape white rot and / or grape anthracnose.

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