A strain of Pseudomonas soybean ZTT6-4 and its application

Abstract

This invention relates to the field of microbial biocontrol, specifically to a strain of *Pseudomonas saccharidus* ZTT6-4 and its applications. This invention discovered a strain of *Pseudomonas saccharidus* (ZTT6-4) through long-term and extensive screening. P. glycinae ZTT6-4, with preservation number CCTCC NO: M 20261057, exhibits excellent inhibitory effects against pepper mild mottle virus, with an average inhibition rate of 65.32%. It can reduce the infection rate of pepper mild mottle virus in crops, alleviate viral disease symptoms, and improve pepper yield and quality. This bacterium can be used to prepare inoculants to inhibit pepper mild mottle virus, showing promising prospects for industrial application.

Description

Technical Field

[0001] This invention relates to the field of microbial biological control, specifically to a strain of Pseudomonas soybean, ZTT6-4, and its applications. Background Technology

[0002] chili( Capsicum annuum Chili peppers (L.) are an important vegetable variety and a major spice crop worldwide. Rich in nutrients, diverse in flavor, and vigorous in growth, chili peppers can be eaten fresh. Besides their nutritional value, they also have significant economic and social value. Pepper mild mottle virus (PMMoV) primarily affects the leaves and fruits of chili pepper plants. Infection causes leaves to turn pale green, develop mosaic patterns, and become mottled; in severe cases, leaves become wrinkled and deformed. Plant growth is inhibited, resulting in significant stunting. Fruits become smaller, develop pale green patches or necrotic spots, and become severely deformed, directly reducing yield and commercial value. This virus can be transmitted through seed-borne infection, sap contact, and agricultural operations, making control difficult and posing a persistent threat to chili pepper producing areas. Currently, there are no highly effective chemical pesticides for plant viruses. Researchers are attempting to screen for antiviral bacteria from natural microorganisms. Researchers have screened various strains and metabolites with antiviral activity from microorganisms and their metabolites, some of which have been widely applied in agricultural production. Currently registered and used microbial agents mainly include Bacillus-based microbial agents, photosynthetic bacteria, ningnanmycin, pyrimethamine, lentinan, and amino oligosaccharides. Although some progress has been made in the microbial control of plant viral diseases, research and application in this field still face many problems such as limited resources, poor efficacy, and short duration of action. Therefore, screening and developing microorganisms and their preparations with resistance to PMMoV has significant theoretical and practical value. Summary of the Invention

[0003] Screening and developing microorganisms with antagonistic effects against plant viruses from soil is an important research direction for researchers in this field. To address the problem of effectively controlling pepper mild mottle virus, this invention, through extensive screening, obtained a strain of *Pseudomonas soybeanii* (…). P. glycinae This invention utilizes the method to control pepper mild mottle virus, thus completing the present invention. The technical solution of the present invention is as follows: In one aspect, this invention discloses a strain of Pseudomonas soybeanii ZTT6-4 ( Pseudomonas glycinae ZTT6-4), this strain has been deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on May 25, 2026, with accession number CCTCC NO: M 20261057.

[0004] In one aspect, the present invention provides a microbial inoculant containing the *Pseudomonas soybeanii* strain ZTT6-4 described in this invention. Pseudomonas glycinae ZTT6-4, P. glycinae ZTT6-4).

[0005] In one embodiment, the microbial agent is selected from liquid formulations, lyophilized powders, or solid fixatives.

[0006] In one embodiment, the liquid preparation comprises a fermentation broth of *Pseudomonas soybeanae*, which is obtained by large-scale fermentation of the *Pseudomonas soybeanae* strain ZTT6-4 of the present invention using a suitable culture medium and culture conditions.

[0007] In one embodiment, the lyophilized powder is prepared by freeze-drying a mixture containing *Pseudomonas soybeanii* ZTT6-4 fermentation broth and a lyophilization protectant. Commonly used lyophilization protectants are selected from one or more of sucrose, glycerol, sodium alginate, and albumin. In this invention, the lyophilization protectant serves to reduce the mortality rate of the strain during freezing, thereby maintaining the activity of the cells.

[0008] In one embodiment, the solid fixative is obtained by immobilizing Pseudomonas soybean spp. ZTT6-4 with an inorganic or organic carrier. Commonly used carriers are selected from one or more of activated carbon, diatomaceous earth, porous ceramics, and synthetic polymer gel carriers.

[0009] In one aspect, the present invention relates to a method for preventing and controlling pepper mild mottle virus by applying ground-grown Pseudomonas soybean 6-4 to objects infected with pepper mild mottle virus.

[0010] In one embodiment, the object is a chili pepper, bell pepper, or tobacco. Those skilled in the art will understand that the natural hosts of PMMoV are primarily Solanaceae crops, especially plants of the genera *Capsicum* and *Nicotiana*.

[0011] The method for preventing and controlling pepper mild mottle virus according to the present invention includes spraying, spraying, and application in combination with other fertilizers or pesticides to the target crop.

[0012] In one aspect, this invention provides the application of *Pseudomonas soybeanii* ZTT6-4 in the control of pepper mild mottle virus (PMMoV). *Pseudomonas soybeanii* ZTT6-4 exhibits significant inhibitory effects on PMMoV, effectively reducing the virus infection rate in pepper plants, alleviating viral disease symptoms, and improving pepper yield and quality.

[0013] Those skilled in the art will understand that, in this invention, "Pseudomonas soybeanii ( P. glycinae"ZTT6-4", "Pseudomonas soybeanii ZTT6-4", "Strain ZTT6-4", P. glycinae ZTT6-4” Pseudomonas glycinae "ZTT6-4" refers to the *Pseudomonas soybeanus* strain with accession number CCTCC NO: M 20261057, and they have the same meaning.

[0014] Beneficial effects (1) Through long-term and extensive screening, this invention discovered a strain of Pseudomonas soybean, ZTT6-4, which has a good inhibitory effect on pepper mild mottle virus, with an average inhibition rate of 65.32%. It can reduce the infection rate of crops to pepper mild mottle virus, alleviate viral disease symptoms, and improve the yield and quality of peppers, and has good industrial application value.

[0015] (2) This invention provides a new means for the prevention and control of pepper mild mottle virus in production. Screening and utilizing natural microorganisms for the prevention and control of plant viruses can promote the construction and innovation of a green prevention and control system for plant viral diseases, and provide strong support for promoting green agricultural development and realizing sustainable agricultural development. Attached Figure Description

[0016] Figure 1 The *Pseudomonas soybeanii* ZTT6-4 strain obtained by screening in this invention (… P. glycinae ZTT6-4) growth status on the surface of NA medium.

[0017] Figure 2 The present invention discloses the inhibitory effect of a strain of Pseudomonas soybean ZTT6-4 on PMMoV. In this invention, A is the control group with PMMoV mixed with liquid NA medium in the left half of the leaf and PMMoV mixed with ZTT6-4 bacterial solution in the right half of the leaf as the treatment group; B is the control group with PMMoV mixed with sterile distilled water in the left half of the leaf and PMMoV mixed with Ningnanmycin bacterial agent in the right half of the leaf as the treatment group. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. Unless otherwise stated, all reagents used in this invention are analytical grade reagents. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0019] Example 1: Screening and Identification of Strains (1) Sampling: In chili fields where viral disease symptoms are severe, select healthy chili plants and take 10-15 cm of their rhizosphere soil.

[0020] (2) Take 10 g of soil sample, add 50 mL of sterile water, and shake thoroughly with glass beads until no large particles remain. Let stand at room temperature for 30 min. Take 1 mL of the supernatant and dilute it at a ratio of 1:10 (e.g., mix 1 mL of the original solution with 9 mL of physiological saline) to prepare 10 g of the original solution. - ¹Diluent. Continue with 10-fold serial dilutions (10... - ²、10 - (³ etc.), for later use.

[0021] (3) Take 100 μL of each dilution and spread it evenly on the surface of NA nutrient agar plates (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) using a sterile pipette. Gently tap the surface of the plate with sterile glass beads to distribute the bacterial solution evenly. Invert the plate and place it in a constant temperature incubator at 28℃ for 24-48 h.

[0022] (4) Single colony picking and purification: Observe the colonies, and after cultivation, pick single colonies with different morphologies (such as shape, edge regularity, colony color, etc.). Use a sterile inoculation loop to pick up a single colony and streak it on a new NA nutrient agar plate for isolation. Incubate at 28℃ with the plate inverted, repeating 2-3 times until a pure strain is obtained. The pure strain can be stored for a long time at -80℃ in a medium containing 25% glycerol NA at a final concentration, or used as a test strain for the half-leaf necrotic spot test.

[0023] (5) Screening: Single colony bacteria were selected and cultured in 100 mL Erlenmeyer flasks containing 20 mL of liquid NA medium (beef extract 3.0 g / L, sucrose 10.0 g / L, peptone 5.0 g / L, yeast extract 1.0 g / L, pH=7.1) at 28℃ and 200 rpm for 12-24 h with shaking to ensure a sufficiently high bacterial concentration, which was then used as a reserve inoculum for fermentation. The PMMoV necrotic host, *Nicotiana scabra* (*Nicotiana scabra*), was selected. Nicotiana tabacum The half-leaf method of inoculation was used to determine the inhibition rate of PMMoV infection by different strains of *S. samsun* var. *Samsun* using the allergic necrosis response of *S. samsun* to PMMoV.

[0024] (6) Identification: The strain ZTT6-4, which had the highest virus inhibition rate obtained in step (5), was selected and identified using conventional microbial identification methods such as colony growth and morphological observation. The ZTT6-4 strain obtained in this screening formed round, opaque, white colonies with smooth protrusions, regular edges, and stickiness on NA medium, and was Gram-negative. Pathogen DNA was extracted using the Omega Bacterial Genomic DNA Purification Kit (D3350-01). Based on the gene encoding the α subunit of RNA polymerase... rpoA Gene-specific primers rpoA-F (5'-ATGCAGAGTTCGGTAAATGAGTTCCTGACC-3', SEQ ID No. 2) and rpoA-R (5'-TCAGGCAGTGGCCTTGTCGTCTTTCTT-3', SEQ ID No. 3) were designed. PCR amplification was performed using TOYOBO KOD FX (KFX-101) under the following conditions: 94℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s; 58℃ annealing for 30 s; 68℃ extension for 1 min 30 s, for 30 cycles; and 68℃ extension for 10 min. The PCR products were recovered using an Omega gel recovery kit (D3350-01), then ligated, transformed, and sequenced using a Hieff Clone Zero TOP0-TACloning Kit (10907ES). The sequencing results are shown in SEQ ID No. 1. P. glycinae of rpoA The gene similarity (Genbank accession number: CP014205.2) reached 99.60%. This bacterium was identified as... P. glycinae The strain was named *Pseudomonas soybeanii* ZTT6-4. It was also deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, on May 25, 2026, with accession number CCTCC M 20261057.

[0025] Example 2: Comparison of PMMoV inhibitory effects of Pseudomonas soybean ZTT6-4 Pseudomonas soybean ZTT6-4 ( P. glycinae Colonies of *Pseudomonas soybeanii* ZTT6-4 were fermented to obtain a bacterial culture. Single colonies of *Pseudomonas soybeanii* ZTT6-4 were then picked and cultured in liquid NA medium at 28°C and 200 rpm for 12-24 h. The OD of the bacterial culture was measured using a spectrophotometer until the culture was fully osmotic. 600 The target concentration was 0.8. Healthy, uniformly growing tobacco plants with 4-6 leaves were selected. Fully infected young leaves of PMMoV were used as the virus source, ground and filtered with phosphate buffer at a 1:10 (mass-to-volume) ratio, and used as the virus test sample. An equal volume of the virus test sample was mixed with liquid NA medium, and after 30 min, the sample was rubbed onto the left half of the leaf with necrotic spots as the control group. An equal volume of ZTT6-4 bacterial suspension was mixed with the virus test sample, and after 30 min, the sample was rubbed onto the right half of the leaf with necrotic spots as the treatment group. Ningnanmycin was used as a positive control. Following the same method, Ningnanmycin was mixed evenly with the virus test sample and applied to the right half of the leaf as the treatment group, while the left half of the leaf was not evenly coated with the bacterial suspension. A mixture of distilled water and the virus test sample was used as the control group. After 48 h, when necrotic spots appeared on the leaf surface, the number of necrotic spots on the left and right halves of the leaf was recorded. The experiment was repeated three times, with three leaves inoculated per tobacco plant. The results are as follows: Figure 2 As shown in Table 1, the number of necrotic spots on the left and right halves of the leaf and the inhibition rate are as follows: ; Table 1: Comparison of the inhibitory effects of different treatments on PMMoV Inhibition rate (%) = [(number of necrotic spots in control group - number of necrotic spots in treatment group) / number of necrotic spots in control group] × 100%, see Table 1. The average inhibition rate was used for evaluation. Strain ZTT6-4 had a significant antagonistic effect on PMMoV. Three repeated experiments showed that strain ZTT6-4 had an average inhibition rate of 65.32% against PMMoV, while ningnanmycin had an average inhibition rate of 39.81% against PMMoV. The Pseudomonas soybean strain ZTT6-4 obtained by screening and comparison in this invention has a significantly higher inhibitory effect on PMMoV than the chemical pesticide ningnanmycin.

[0026] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A strain of *Pseudomonas soybeanii* ZTT6-4, characterized in that, The strain has been deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China, on May 25, 2026, with accession number CCTCC NO: M 20261057.

2. A microbial inoculant, characterized in that, The bacterial agent contains the *Pseudomonas soybeanii* ZTT6-4 strain as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The microbial agent is selected from one of the following: liquid preparation, lyophilized powder, or solid fixative.

4. The microbial agent according to claim 3, characterized in that, The liquid preparation contains fermentation broth of Pseudomonas soybeanii ZTT6-4.

5. The microbial agent according to claim 3, characterized in that, The freeze-dried powder is prepared by freeze-drying a mixture containing Pseudomonas soybean ZTT6-4 fermentation broth and a freeze-drying protectant.

6. The microbial agent according to claim 5, characterized in that, The freeze-drying protectant is selected from one or more of sucrose, glycerol, sodium alginate, and albumin.

7. The microbial agent according to claim 3, characterized in that, The solid fixative is obtained by immobilizing and binding Pseudomonas soybeanus ZTT6-4 with an inorganic or organic carrier.

8. A method for preventing and controlling pepper mild mottle virus, characterized in that, Apply the soybean Pseudomonas ZTT6-4 as described in claim 1 to a subject infected with pepper mild mottle virus.

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

10. The application of the *Pseudomonas soybeanii* ZTT6-4 according to claim 1 in the prevention and control of pepper mild mottle virus.

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