Application of Bacillus cereus in the prevention and control of pepper mild mottle virus

Abstract

The present invention belongs to the technical field of microbial biological control, and specifically relates to a Bacillus cereus having a control effect on pepper mild mottle virus and an application thereof. Bacillus cereus DZ1) has been registered and preserved in the China Center for Type Culture Collection (CCTCC) with a preservation date of September 11, 2023; the preservation number is CCTCC NO: M20231663. Its average inhibition rate against PMMoV reached 93.8%. The screening and use of natural microbial antagonists to control pepper mild mottle virus meets the national green prevention and control development needs, reduces the pollution of chemical pesticides to the soil and environment, and avoids the development of drug resistance in plant pathogens.

Description

Technical Field

[0001] The invention relates to the field of microbial biological control, and in particular to a strain Bacillus cereus capable of controlling pepper mild mottle virus and an application thereof. Background Art

[0002] Pepper mild mottle virus PMMoV belongs to the Virgaviridae family and is an important viral disease that threatens pepper production worldwide. It causes dwarfing and slow growth of plants, mottled leaves, mosaic, chlorosis, deformity, and uneven fruit coloring, resulting in serious yield losses. In addition, PMMoV can enter the human body with diseased peppers, and after being digested and excreted from the human body, it still has infectious activity. It was first reported in my country in Shihezi, Xinjiang in 1994. Because PMMoV can be transmitted through friction of juice and through seeds, it is now widely distributed in my country. At present, laboratory studies have also shown that pepper mild mottle virus can also locally infect plants such as common tobacco, heartleaf tobacco, and dead spot three-born tobacco.

[0003] Microbial pesticides are recognized as non-polluting pesticides with the characteristics of abundant resources, low cost and no pollution to the environment. At the same time, the widespread use of microbial agents can effectively reduce the use of organic and inorganic pesticides by humans, alleviate the serious environmental pollution and ecological balance caused by traditional pesticides. In the prevention and control of PMMoV, some microorganisms also show obvious effects. For example, Bacillus megaterium and Ralstonia pickettii can effectively inhibit the soil-borne harm of PMMoV. Pseudomonas oleovorans strain KBPF-004 can inhibit the mechanical transmission of the virus. It can be seen that microbial pesticides can bring new ideas and means to the prevention and control of PMMoV. Therefore, screening and developing more strains that can inhibit PMMoV is of great significance for the prevention and control of the virus. Summary of the invention

[0004] Providing more effective strains that can inhibit pepper mild mottle virus is also a direction that those skilled in the art continue to study. Based on this, through a large number of screening and testing, the present invention has found a cereus spore strain that can have a good inhibitory effect on pepper mild mottle virus, thereby completing the present invention.

[0005] In one aspect of the present invention, the present invention provides a Bacillus cereus having a preventive and therapeutic effect on pepper mild mottle virus, wherein the Bacillus cereus is Bacillus cereus DZ1 (Bacillus cereus DZ1), which has been registered and preserved in the China Center for Type Culture Collection (CCTCC), the preservation address is Wuhan University, Wuhan, China, and the preservation date is September 11, 2023; the preservation number is CCTCC NO: M20231663.

[0006] In one aspect of the present invention, the present invention provides a microbial agent, wherein the agent contains the Bacillus cereus DZ1 described in the present invention.

[0007] In the present invention, the bacterial agent is one of a liquid preparation, a freeze-dried powder, and a solid fixative. The liquid preparation contains a fermentation broth of Bacillus cereus. That is, the fermentation broth containing bacteria is obtained by large-scale fermentation of the Bacillus cereus DZ1 strain of the present invention through appropriate culture medium and culture conditions.

[0008] In the present invention, the lyophilized powder is prepared by freeze-drying the fermentation liquid containing Bacillus cereus and a lyophilization protective agent. Common cryoprotectants such as sucrose, glycerol, sodium alginate, and albumin can be used. The role of the cryoprotectant is to ensure or reduce the mortality rate of the strain during the freezing process, thereby improving the activity of the bacteria.

[0009] In the present invention, the solid fixative is prepared by fixing Bacillus cereus on a carrier. Common carriers include porous silica, a mixed embedding carrier of PVA and sodium alginate, and the like.

[0010] In one aspect of the present invention, the present invention discloses the use of a microbial agent in preventing and controlling pepper mild mottle virus, wherein the microbial agent contains the Bacillus cereus DZ1 according to claim 1.

[0011] In one aspect of the present invention, a method for preventing and controlling pepper mild mottle virus is disclosed, wherein Bacillus cereus DZ1 is applied to an object infected with pepper mild mottle virus, wherein the object is pepper, sweet pepper or tobacco.

[0012] In the present invention, the control method can be various, including spraying, sprinkling, and applying with other fertilizers or pesticides to the target plants.

[0013] In one aspect of the present invention, the present invention also provides a Bacillus cereus culture, which is obtained by fermenting and culturing the Bacillus cereus DZ1 (Bacillus cereus DZ1) of the present invention. The preparation of a large amount of culture lays a foundation for the preparation of microbial pesticides.

[0014] Beneficial Effects

[0015] The present invention obtains new strains capable of inhibiting pepper mild mottle virus through screening, and tests show that the average inhibition rate of the Bacillus cereus of the present invention on pepper mild mottle virus reaches 93.8%, which provides new options and ideas for the prevention and treatment of pepper mild mottle virus, screens and utilizes natural microbial antagonistic bacteria to prevent and treat pepper mild mottle virus, reduces the pollution of chemical pesticides to soil and the environment, and avoids the development of drug resistance by plant pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The present invention screens and obtains Bacillus cereus DZ1 (Bacillus cereus DZ1).

[0017] Figure 2 The inactivation effect of Bacillus cereus DZ1 and the conventional antiviral agent Ningnanmycin on PMMoV, wherein A is the inactivation of PMMoV virus by antagonistic strain DZ1 (right side of leaf) and negative control (left side of leaf, only containing virus but not containing agent); B is the inactivation of PMMoV by Ningnanmycin (right side of leaf) and negative control (left side of leaf, only containing virus but not containing agent) (the picture is the front of tobacco). C is the inactivation of PMMoV virus by Bacillus amyloliquefaciens (right side of leaf) and negative control (left side of leaf, only containing virus but not containing agent). DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with specific embodiments. The technical solutions of the present invention are conventional methods in the art unless otherwise specified, and the reagents or materials are conventional reagents and are sourced from commercial channels unless otherwise specified.

[0019] Example 1

[0020] Screening and identification of bacterial strains

[0021] (1) Sampling: Select healthy tobacco plants from tobacco fields where tobacco mosaic disease has occurred, and select rhizosphere soil from 20 cm below the roots of the tobacco plants for later use;

[0022] (2) Grinding: Place 10 g of the reserved soil in a sterilized Erlenmeyer flask, add 20 ml of sterile water, shake until the liquid becomes turbid, and leave it at room temperature for 10 minutes for later use;

[0023] (3) Purification: 200 μL of the liquid supernatant after quiescence was sucked up with a sterile pipette tip, and the plate method was used on NA medium (beef extract 3.0 g / L, sucrose 10.0 g / L, peptone 5.0 g / L, yeast extract 1.0 g / L, agar 15.0 g / L, pH = 7.1), and cultured at 28°C for 24 h. After the colonies grew on the separation plate, the isolated single colonies were picked out one by one with an inoculation loop according to the characteristics of the microbial colonies, such as morphology, size, surface structure, edge structure, texture, gloss, transparency, color and soluble pigment produced. The single colonies with different morphologies were picked out and streaked on the corresponding blank NA medium plates for purification, and then inverted and cultured in a constant temperature incubator at 28°C for 24 h to obtain the purified strains for later use;

[0024] (4) Screening: Select a single bacterial colony and place it in a sterilized 100 ml conical flask containing 20 ml of culture medium, shake and culture (28°C, 200 r / min) for 24 h to make the bacterial liquid content large enough for fermentation. Select PMMoV's necrotic host, Nicotiana glutinosa, and use the half-leaf method for friction inoculation test: Using the allergic necrosis reaction of Nicotiana glutinosa to PMMoV, the half-leaf method was used to determine the inhibition rate of PMMoV infection by different strains.

[0025] (5) Identification: The strain DZ1 with the highest inhibition rate in step (4) was selected for physiological, biochemical and molecular biological identification according to conventional microbial identification methods. The strain DZ1 obtained in this screening was determined to have an off-white colony, opaque, rough surface, and molten wax-like appearance. It was Gram-positive (see Figure 1

[0026] Example 2

[0027] Comparison of PMMoV Inhibition Effects of Bacillus cereus DZ1

[0028] The colony of Bacillus cereus DZ1 was fermented to obtain bacterial liquid. The half-leaf method was used to determine the inhibition rate of Bacillus cereus DZ1 on PMMoV infection by using the allergic necrosis reaction of spot heart leaf tobacco to PMMoV. The young leaves of PMMoV virus source with full disease were ground and filtered with distilled water at a ratio of 1:10 (mass to volume) and used as virus test samples. The virus test sample was mixed with equal volumes of liquid NA culture medium, and rubbed on the left half of the spot heart leaf tobacco after 10 minutes as a control. Bacillus cereus DZ1 was mixed with equal volumes of virus samples, and rubbed and inoculated on the right half of the spot heart leaf tobacco after 10 minutes as treatment group 1; in addition, the Ningnanmycin treatment group was used as a positive control, and Bacillus amyloliquefaciens was used as treatment group 2. After the spot appeared, the number of spots on the left and right halves was recorded respectively. Three replicates were performed, and three leaves were inoculated for each tobacco plant. The number of dead spots on the left and right half leaves and the inhibition rate are shown in Table 1:

[0029]

[0030] Table 1 Comparison of the inhibitory effects of different treatments on PMMoV

[0031] Inhibition rate (%) = [(number of control necrosis spots - number of treated necrosis spots) / number of control necrosis spots] × 100%.

[0032] Referring to Table 1, the strain DZ1 has a better antagonistic effect on PMMoV as evaluated by the average inhibition rate. Three repeated tests show that the inhibition rate of DZ1 fermentation broth on PMMoV can reach 93.8%, while the average inhibition rate of Ningnanmycin is only 51.1%. The PMMoV inhibition effect of Bacillus cereus DZ1 screened by the present invention is significantly higher than that of the chemical pesticide Ningnanmycin and Bacillus amyloliquefaciens.

[0033] The above content is a further detailed description of the present invention in combination with specific implementation methods. It cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the scope of protection determined by the claims submitted for the present invention.

Claims

1. A Bacillus cereus having a preventive and therapeutic effect on pepper mild mottle virus, characterized in that: The Bacillus cereus is Bacillus cereus DZ1( Bacillus cereus DZ1), which has been registered and deposited in the China Center for Type Culture Collection (CCTCC), with the deposit address being Wuhan University, Wuhan, China, and the deposit date being September 11, 2023; the deposit number is CCTCC NO: M20231663.

2. A microbial agent, characterized in that: The bacterial agent contains the Bacillus cereus DZ1( Bacillus cereus DZ1).

3. The bacterial agent according to claim 2, characterized in that The bacterial agent is one of a liquid preparation, a freeze-dried powder, and a solid fixative.

4. The bacterial agent according to claim 3, characterized in that The liquid preparation contains Bacillus cereus fermentation liquid.

5. The bacterial agent according to claim 3, characterized in that The freeze-dried powder is prepared by freeze-drying a fermentation liquid containing Bacillus cereus and a freeze-drying protective agent.

6. The bacterial agent according to claim 3, characterized in that The solid fixing agent is prepared by fixing Bacillus cereus on a carrier.

7. Use of a microbial agent in preventing and controlling pepper mild mottle virus, characterized in that: The bacterial agent contains the Bacillus cereus DZ1( Bacillus cereus DZ1).

8. A method for preventing and controlling pepper mild mottle virus, characterized in that: administering to a subject infected with pepper mild mottle virus the Bacillus cereus DZ1 ( Bacillus cereus DZ1).

9. The method according to claim 8, characterized in that The object is chili pepper, sweet pepper or tobacco.

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