Application of Stenotrophomonas maltophilia in the prevention and control of pepper mild mottle virus

Abstract

The present invention belongs to the technical field of plant protection microbial biological control, and specifically, the present invention relates to a Stenotrophomonas maltophilia having a control effect on pepper mild mottle virus (PMMoV) and an application thereof. Stenotrophomonas maltophilia DZ4) has been registered and preserved in the China Center for Type Culture Collection (CCTCC) on September 11, 2023; the preservation number is CCTCC NO: M20231664, and its average inhibition rate for PMMoV reached 93.0%. The present invention can significantly reduce the use of chemical pesticides by screening and using natural microbial antagonists to control pepper mild mottle virus, alleviate the pollution of chemical pesticides to soil and the environment, and avoid the generation 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 Stenotrophomonas maltophilia with pepper mild mottle virus control function and an application thereof. Background Art

[0002] Pepper mild mottle virus (PMMoV) is a positive single-stranded RNA virus that can cause leaf mottle, mosaic, chlorosis, deformity, uneven fruit coloring, plant dwarfing and slow growth, resulting in serious yield losses. The positive detection rate of commercial seeds sold in the Beijing market reached 61.11%. PMMoV can be spread not only through friction with juice, but also through seeds. It is an important viral disease threatening pepper production worldwide.

[0003] Microbial pesticides are rich in resources, low in cost, and do not pollute the environment. The widespread use of microbial pesticides can effectively reduce the use of organic and inorganic pesticides by humans, and alleviate the environmental pollution and ecological balance caused by traditional pesticides. Some microorganisms also show obvious effects in the prevention and control of pepper mild mottle virus, among which 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. CN202310654411.1 discloses that an extreme oriental Pseudomonas has the ability to prevent and control pepper mild mottle virus; CN202110559463.1 discloses that a strain of extremely fine Alternaria can produce Taishanmycin, which can prevent and control a variety of plant viruses, including pepper mild mottle virus. It can be seen that microbial pesticides can bring new ideas and means for the prevention and control of PMMoV. Therefore, screening and developing more strains that can inhibit PMMoV is of great significance for the prevention and treatment of the virus. Summary of the invention

[0004] Providing more effective strains that can inhibit pepper mild mottle virus is also a direction that technicians in this field continue to study. Based on this, through a large number of screening and tests, the present invention discovered a new strain of Stenotrophomonas maltophilia that can prevent and treat pepper mild mottle virus, thereby completing the present invention.

[0005] The present invention provides the following technical solutions:

[0006] The present invention provides a Stenotrophomonas maltophilia, wherein the Stenotrophomonas maltophilia DZ4 (Stenotrophomonas maltophilia DZ4) has been registered and preserved in the China Center for Type Culture Collection (CCTCC), the preservation address is Wuhan University, Wuhan, China, the preservation date is September 11, 2023, and the preservation number is CCTCCNO: M 20231664.

[0007] In one aspect of the present invention, the present invention also provides the use of the Stenotrophomonas maltophilia and its metabolites in the prevention and control of pepper mild mottle virus. The prevention and control methods can be various, including spraying, sprinkling, and applying to the target plants together with other fertilizers or pesticides.

[0008] In one aspect of the present invention, the present invention also provides a microbial agent, wherein the agent contains the Stenotrophomonas maltophilia or its metabolites in the present invention. Stenotrophomonas maltophilia can be cultured on a large scale by large-scale fermentation for the preparation of the agent. The specific fermentation conditions can be adjusted by those skilled in the art according to the growth characteristics of the bacteria, such as using a culture medium adapted to the growth of the bacteria, and controlling the temperature, oxygen, pH value, rotation speed and other conditions during the fermentation process. The metabolites described in the present invention are obtained by separating the supernatant obtained by fermenting Stenotrophomonas maltophilia, i.e., the fermentation culture supernatant. It is well known to those skilled in the art that the bacteria will produce a variety of metabolites during the fermentation process, some of which are released into the fermentation broth through secretion or after the bacteria die and break. Therefore, it is often believed that these fermentation supernatants also have the same effect as the fermentation broth containing the bacteria. In the present invention, the separation method of the metabolites includes but is not limited to centrifugation, filtration, suction filtration, chromatography, and resin separation.

[0009] In one aspect of the present invention, the microbial agent prepared by the present invention can be used for the prevention and treatment of pepper mild mottle virus. The microbial agent can also be prepared in various forms according to demand and convenience of use. As long as the preparation method and preparation form of the existing microbial pesticide can be used for the preparation of the agent of the present invention. In the present invention, the agent can be a liquid preparation, a lyophilized powder, or a solid fixative. The liquid preparation can be prepared into various suspensions, emulsions, and dispersants; the lyophilized powder is a commonly used preparation form of microbial pesticides, which can better protect the activity of microorganisms. The lyophilized powder can also contain a variety of substances that have a protective or stabilizing effect on the activity of microorganisms, such as glycerol, protein, sugar alcohol, DMSO, and buffer; the solid preparation fixes the microorganism or its metabolites on a fixed carrier through various forms such as adsorption and connection, and can also maintain the activity of the microorganism for a certain period of time, playing an enrichment and synergistic role.

[0010] thus,

[0011] The invention discloses a liquid bacterial agent, which is prepared by fermenting the Stenotrophomonas maltophilia of the invention.

[0012] The invention discloses a freeze-dried powder, which is prepared by fermenting the Stenotrophomonas maltophilia of the invention and then freeze-drying it. The invention discloses a solid bacterial agent, which is prepared by fermenting the Stenotrophomonas maltophilia of the invention and then fixing it on a fixed carrier.

[0013] In one aspect of the present invention, the present invention also provides a method for preventing and controlling pepper mild mottle virus, wherein the bacterial agent of the present invention is applied to a target plant infected or potentially infected with pepper mild mottle virus. Preferably, the target plant is pepper, sweet pepper, or tobacco.

[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 Stenotrophomonas maltophilia for pepper mild mottle virus reaches 93.0%, which provides new options and ideas for the prevention and control of pepper mild mottle virus. The screening and use of natural microbial antagonistic bacteria to prevent and control pepper mild mottle virus meets the national green prevention and control development needs, 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 screened and obtained Stenotrophomonas maltophilia DZ4.

[0017] Figure 2 The inactivation effect of Stenotrophomonas maltophilia DZ4 and the conventional antiviral agent Ningnanmycin on PMMoV, wherein A is the result of Stenotrophomonas maltophilia DZ4 inactivating PMMoV virus (right side of leaf) and negative control (left side of leaf, only PMMoV, without Stenotrophomonas maltophilia DZ4); B is the result of Ningnanmycin inactivating PMMoV virus (right side of leaf) and negative control (left side of leaf, only PMMoV, without Ningnanmycin) (the picture is the front of tobacco). C is the result of Pseudomonas fluorescens inactivating PMMoV virus (right side of leaf) and negative control (left side of leaf, only PMMoV, without Pseudomonas fluorescens). 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 ensure that the bacterial liquid content is large enough for fermentation. Select the PMMoV dead spot host Nicotiana glutinosa and use the half-leaf method for friction inoculation test: Using the hypersensitive 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 DZ4 with the highest inhibition rate in step (4) was selected for physiological, biochemical and molecular biological identification according to conventional microbial identification methods. After observation and measurement, the strain DZ4 obtained in this screening had round, smooth, moist, light yellow colonies and Gram-negative staining. The pathogen DNA was extracted using the Omega genomic DNA purification kit (D3350-01). The 16S rDNA genes 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were used. The polymerase m5 was used for superluminal PCR amplification was performed under the following conditions: 95°C pre-denaturation for 3 min; 94°C denaturation for 25 s, 60°C annealing for 25 s, 72°C extension for 1 min 30 s, 30 cycles; 72°C extension for 10 min. The PCR product was recovered using the Omega kit (D3350-01) and then purified using Yisheng

[0026] (Stenotrophomonas maltophilia DZ4), the strain has been registered and deposited in the China Center for Type Culture Collection (CCTCC), the deposit address is Wuhan University, Wuhan, China, the deposit date is September 11, 2023; the deposit number is CCTCC NO: 20231664.

[0027] Example 2

[0028] Comparison of PMMoV Inhibition Effects of Stenotrophomonas maltophilia DZ4

[0029] The colony of Stenotrophomonas maltophilia DZ4 was fermented to obtain bacterial liquid. The half-leaf method was used to determine the inhibition rate of Stenotrophomonas maltophilia DZ4 on PMMoV infection by using the allergic necrosis reaction of heartleaf 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 an equal volume of liquid NA culture medium, and rubbed on the left half of the heartleaf tobacco after 10 minutes as a control. Stenotrophomonas maltophilia DZ4 was mixed with an equal volume of virus sample, and rubbed and inoculated on the right half of the heartleaf tobacco after 10 minutes as treatment group 1; in addition, the Ningnanmycin treatment group was used as a positive control, and Pseudomonas fluorescens was used as treatment group 2. After the necrosis spots appeared, the number of necrosis spots on the left and right halves were 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:

[0030]

[0031]

[0032] Table 1 Comparison of the inhibitory effects of different treatments on PMMoV

[0033] Inhibition rate (%) = [(number of control necrosis spots - number of treated necrosis spots) / number of control necrosis spots] × 100%.

[0034] Referring to Table 1, based on the average inhibition rate, Stenotrophomonas maltophilia DZ4 has a better antagonistic effect on PMMoV. Three repeated tests showed that the inhibition rate of the fermentation broth of Stenotrophomonas maltophilia DZ4 on PMMoV can reach 93.0%, while the average inhibition rate of Ningnanmycin is only 61.1%. The inhibitory effect of Stenotrophomonas maltophilia DZ4 screened by the present invention on PMMoV is significantly higher than that of the chemical pesticide Ningnanmycin and Pseudomonas fluorescens.

[0035] 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 Stenotrophomonas maltophilia, characterized in that The Stenotrophomonas maltophilia is Stenotrophomonas maltophilia DZ4 ( Stenotrophomonas maltophilia )DZ4, the deposit number is CCTCC NO: M20231664, deposited in China Center for Type Culture Collection (CCTCC), the deposit address is Wuhan University, Wuhan, China, and the deposit date is September 11, 2023.

2. Use of the Stenotrophomonas maltophilia according to claim 1 in preventing and controlling pepper mild mottle virus.

3. A microbial agent, characterized in that: The bacterial agent contains the Stenotrophomonas maltophilia described in claim 1.

4. Use of the bacterial agent according to claim 3 in preventing and controlling pepper mild mottle virus.

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

6. The bacterial agent according to claim 5, characterized in that The liquid preparation is prepared by fermenting the Stenotrophomonas maltophilia according to claim 1.

7. The bacterial agent according to claim 5, characterized in that The freeze-dried powder is prepared by fermenting the Stenotrophomonas maltophilia according to claim 1 and then freeze-drying it.

8. The bacterial agent according to claim 5, characterized in that The solid bacterial agent is prepared by fermenting the Stenotrophomonas maltophilia described in claim 1 and fixing it on a fixed carrier.

9. A method for preventing and controlling pepper mild mottle virus, characterized in that: A subject infected with pepper mild mottle virus is administered with the Stenotrophomonas maltophilia according to claim 1 or a fermentation supernatant thereof.

10. The method according to claim 9, characterized in that The objects are chili peppers, sweet peppers, and tobacco.

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