Pseudomonas aeruginosa and application thereof

Abstract

The present application belongs to the field of agricultural microorganism technology, and particularly relates to a Pseudomonas aeruginosa strain and application thereof Pseudomonas aeruginosa R5‑2, and the strain number is CGMCC No.34796.The strain can secrete iron carrier, chitinase and other active substances, and can effectively inhibit the growth of multiple pathogenic fungi such as Fusarium oxysporum, Verticillium dahliae, Rhizoctonia solani and Fusarium graminearum.The experiment shows that the fungicide containing the strain has a significant prevention and treatment effect on the root rot of wolfberry caused by Fusarium oxysporum and the tomato wilt.The present application provides a new, environment-friendly microbial resource for preventing and treating soil-borne fungal diseases of wolfberry, tomato and other crops, and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbiology technology, specifically relating to a strain of Pseudomonas aeruginosa and its applications. Background Technology

[0002] Goji berries are a valuable crop used for both food and medicine. With the expansion of intensive cultivation, soil-borne diseases, especially goji berry root rot, have become increasingly serious. This disease is mainly caused by a combination of pathogenic fungi, including Fusarium oxysporum, Fusarium solani, and Rhizoctonia solani, which can lead to root rot, vascular bundle browning, and subsequently yellowing, wilting, and even death of the above-ground parts. This often results in huge yield losses and seriously restricts the sustainable development of the industry.

[0003] Currently, the control of root rot in wolfberry mainly relies on chemical fungicides, such as carbendazim, thiophanate-methyl, and hymexazol. Although chemical control can achieve certain results in the short term, long-term and excessive use can easily lead to problems such as pathogen resistance, pesticide residues, and damage to the soil ecosystem, posing potential risks to the quality and safety of wolfberry. Therefore, seeking new, efficient, safe, and environmentally friendly methods for controlling root rot in wolfberry has become an urgent need for the healthy development of the wolfberry industry.

[0004] Biological control, as an environmentally friendly alternative or supplementary strategy, has received widespread attention in recent years. Utilizing beneficial microorganisms and their metabolites to inhibit plant pathogens is an important approach to achieving green and sustainable agriculture. *Pseudomonas aeruginosa* can produce various antimicrobial substances (such as phenazines, hydrogen cyanide, and siderophores) and cell wall-degrading enzymes (such as chitinase), and has a strong rhizosphere colonization ability, showing significant potential in controlling soil-borne crop diseases. However, existing research has largely focused on crops such as wheat, rice, tomato, and cucumber, and research and development of biocontrol strains for *Lycium barbarum* root rot remains lacking. Furthermore, the efficacy of microbial control is greatly affected by ecological adaptability; therefore, screening for locally dominant antagonistic strains from the *Lycium barbarum* rhizosphere is crucial for improving the stability of control efficacy.

[0005] Furthermore, due to the high ecological specificity of microbial strains, isolating and screening locally dominant strains from specific ecological environments (such as healthy wolfberry rhizosphere soil) that exhibit highly efficient antagonistic activity against the pathogen causing wolfberry root rot and are well adapted to the wolfberry rhizosphere environment is crucial for improving the stability and field efficacy of biological control. This not only provides new and effective microbial resources for the green control of wolfberry root rot and enriches the strain pool for biological control of wolfberry diseases, but also has significant implications for reducing the use of chemical pesticides, ensuring the safety of wolfberry products and the ecological environment, and promoting the sustainable development of the wolfberry industry. Summary of the Invention

[0006] To solve the above-mentioned technical problems, this invention isolates and screens a strain of *Pseudomonas aeruginosa* from rhizosphere soil samples of wolfberry. Pseudomonas aeruginosaR5-2, through plate confrontation experiments, was found to have antagonistic effects against pathogens *Fusarium oxysporum*, *Verticillium dahliae*, *Rhizoctonia solani*, and *Fusarium graminearum*. The ability of this strain to produce siderophores, β-1,3-glucanase, cellulase, lipase, protease, and chitinase was determined using the perforation method. Pot experiments verified that this strain has significant control efficacy against *Fusarium oxysporum* on wolfberry and tomato, providing an important strain resource for the control of wolfberry root rot and tomato wilt, and solving the technical problem of lacking strains specifically for the control of wolfberry root rot in existing technologies.

[0007] On the one hand, the present invention provides a strain of Pseudomonas aeruginosa. Pseudomonas aeruginosa R5-2 (abbreviated as R5-2 strain) is an endophytic fungus in the root system of wolfberry from Dulan County, Haixi Mongolian and Tibetan Autonomous Prefecture, Qinghai Province. Its preservation information is as follows.

[0008] Strain name: R5-2;

[0009] Classification name: Pseudomonas aeruginosa Pseudomonas aeruginosa ;

[0010] Preservation period: July 4, 2025;

[0011] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections;

[0012] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing;

[0013] Accession number: CGMCC No.34796.

[0014] Furthermore, the 16S rDNA sequence of the *Pseudomonas aeruginosa* is shown in SEQ ID NO:1.

[0015] Furthermore, the *Pseudomonas aeruginosa* can produce siderophores, β-1,3-glucanase, cellulase, lipase, protease, and chitinase.

[0016] Secondly, the present invention also provides a microbial agent containing the Pseudomonas aeruginosa described in the present invention, wherein the active ingredient of the microbial agent is selected from one or more of the Pseudomonas aeruginosa bacterial suspension, fermentation broth, and fermentation broth supernatant.

[0017] Thirdly, a method for preparing the bacterial agent of the present invention is also provided, the method comprising culturing the Pseudomonas aeruginosa of the present invention and collecting the bacterial cells, fermentation broth or fermentation broth supernatant of the Pseudomonas aeruginosa.

[0018] Fourthly, the present invention also provides the application of the Pseudomonas aeruginosa or its agent in inhibiting the growth of pathogens, wherein the pathogens are Fusarium oxysporum, Verticillium dahliae, Rhizoctonia solani, or Fusarium graminearum.

[0019] Fifthly, the invention also provides the application of the *Pseudomonas aeruginosa* or its agent in the prevention and control of root rot in wolfberry, wherein the root rot is induced by *Fusarium oxysporum*.

[0020] Sixthly, the invention also provides the application of the *Pseudomonas aeruginosa* or its agent in the control of tomato wilt, wherein the tomato wilt is induced by *Fusarium oxysporum*.

[0021] Finally, a method for preventing and controlling soil-borne fungal diseases of plants is also provided, the method comprising applying the fungal agent of the present invention to the rhizosphere of a plant, wherein the plant is wolfberry or tomato, and the soil-borne fungal disease is induced by Fusarium oxysporum.

[0022] Furthermore, in the method, the application is carried out by irrigation.

[0023] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0024] (1) This invention isolates a new strain of Pseudomonas aeruginosa from the rhizosphere of Lycium barbarum. Pseudomonas aeruginosa R5-2, this strain has inhibitory effects on Fusarium oxysporum, Verticillium dahliae, Rhizoctonia solani, and Fusarium graminearum, especially exhibiting extremely strong inhibitory activity against Fusarium oxysporum. It provides a new and highly efficient microbial strain resource for the prevention and control of root rot in wolfberry, filling the gap in dedicated biocontrol strains in this field.

[0025] (2) The Pseudomonas aeruginosa provided by the present invention Pseudomonas aeruginosa R5-2 can simultaneously produce siderophores that compete for nutrients and β-cell wall degrading agents. 1,3 It contains multiple antibacterial substances such as glucanase and chitinase, possessing a multi-layered biocontrol mechanism. Pot experiments have confirmed its significant control effect against Fusarium oxysporum disease in wolfberry and tomato.

[0026] (3) The use of this strain and its agent can reduce dependence on chemical pesticides, reduce the risk of residues and resistance, and is environmentally friendly. At the same time, this strain has shown antibacterial activity against a variety of important soil-borne pathogens, showing broad-spectrum biocontrol application potential.

[0027] (4) The strain was isolated from the rhizosphere of wolfberry. As a local microorganism, it may be more adaptable to colonization and function in the rhizosphere environment of wolfberry, which is conducive to improving the stability and effectiveness of biological control in the field. Attached Figure Description

[0028] Figure 1 It is Pseudomonas aeruginosa Pseudomonas aeruginosa The morphology of R5-2 inside the dish.

[0029] Figure 2 It is Pseudomonas aeruginosa Pseudomonas aeruginosa Morphology of strain R5-2 after Gram staining.

[0030] Figure 3 It is Pseudomonas aeruginosa Pseudomonas aeruginosa Phylogenetic tree of R5-2.

[0031] Figure 4 It is Pseudomonas aeruginosa Pseudomonas aeruginosa The graph shows the inhibition rate of R5-2 fermentation broth supernatant against Fusarium oxysporum. A represents different amounts of Pseudomonas aeruginosa added. Pseudomonas aeruginosa The colony growth phenotype of *Fusarium oxysporum* under R5-2 fermentation broth supernatant treatment; B represents different amounts of *Pseudomonas aeruginosa*. Pseudomonas aeruginosa The inhibition rate of R5-2 fermentation broth supernatant against Fusarium oxysporum.

[0032] Figure 5 It is Pseudomonas aeruginosa Pseudomonas aeruginosa R5-2's antibacterial spectrum against different pathogens.

[0033] Figure 6 It is Pseudomonas aeruginosa Pseudomonas aeruginosa The graph shows the test results of R5-2's ability to produce siderophores, β-1,3-glucanase, cellulase, lipase, protease, and chitinase.

[0034] Figure 7 It is Pseudomonas aeruginosa Pseudomonas aeruginosa The effect of R5-2 on controlling Fusarium oxysporum in indoor potted plants on wolfberry.

[0035] Figure 8 It is Pseudomonas aeruginosa Pseudomonas aeruginosa The effect of R5-2 on controlling Fusarium oxysporum in indoor potted plants on tomatoes. Detailed Implementation

[0036] The technical solution of the present invention will be described below with reference to the embodiments. However, the present invention is not limited to the following embodiments.

[0037] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention.

[0038] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0039] Example 1

[0040] This example illustrates the isolation, purification, and identification process of strain R5-2.

[0041] 1. Isolation and purification of bacterial strains

[0042] Roots from healthy wolfberry plants were collected, strictly disinfected, and then ground under aseptic conditions to obtain a root tissue homogenate. The homogenate was spread onto LB agar plates using a dilution-spreading method and incubated at 28°C for 1-3 days. After clearly morphologically distinct single colonies grew on the plates, they were initially screened based on colony morphology, color, and other characteristics. Target single colonies were then streaked onto new LB agar plates for purification. This process was repeated 2-3 times until the desired homogenate was obtained. Figure 1 The pure culture shown is defined as the primary screening strain. The primary screening strain was subjected to plate confrontation with *Fusarium oxysporum*, repeated three times, to screen for strains exhibiting significant antagonistic activity against *Fusarium oxysporum*. A strain exhibiting significant antagonistic activity against *Fusarium oxysporum* was obtained and named R5-2, which was stored at -80°C.

[0043] 2. Gram staining of strain R5-2

[0044] The R5-2 strain, stored at -80℃, was activated on LB agar plates. After 2 days, single colonies were selected to prepare bacterial smears. The smears were fixed and then sequentially stained with crystal violet for 1 min, mordanted with Lugol's iodine for 1 min, destained with 95% ethanol until colorless, and counterstained with safranin for 1 min. After drying, the staining results were observed under an optical microscope. The results are as follows: Figure 2 As shown, strain R5-2 stains red with Gram stain, indicating it is a Gram-negative bacillus.

[0045] 3. Construct a phylogenetic tree for strain R5-2.

[0046] Genomic DNA was extracted from strain R5-2 and used as a template for PCR amplification using the universal primer 27F for the bacterial 16S rDNA gene. The PCR products were detected by agarose gel electrophoresis and then sent to a sequencing company for bidirectional sequencing.

[0047] The sequences of primers 27F and 1492R are as follows:

[0048] 27F (SEQ ID NO:2): 5'-AGAGTTTGATCCTGGCTCAG-3';

[0049] 1492R (SEQ ID NO:3): 5'-GGTTACCTTGTTACGACTT-3'.

[0050] The 16S rDNA sequence of strain R5-2, as shown in SEQ ID NO:1, was subjected to homology comparison using the BLAST program in the NCBI database, and sequences of strains with high similarity were downloaded. A phylogenetic tree was constructed using MEGA 11 software via neighbor-joining. The constructed phylogenetic tree is shown below. Figure 3 As shown, strain R5-2 and Pseudomonas aeruginosa ( Pseudomonas aeruginosa They clustered together, and based on their morphological characteristics and Gram staining, they were identified as *Pseudomonas aeruginosa*. Pseudomonas aeruginosa R5-2.

[0051] Example 2

[0052] This example uses *Pseudomonas aeruginosa*. Pseudomonas aeruginosa In-plate antibacterial activity test of R5-2.

[0053] 1. Determination of Pseudomonas aeruginosa Pseudomonas aeruginosa R5-2 inhibition rate against Fusarium oxysporum

[0054] Determination of Pseudomonas aeruginosa using the plate confrontation culture method Pseudomonas aeruginosa The supernatant of R5-2 fermentation broth showed inhibitory activity against *Fusarium oxysporum*, the pathogen causing root rot in wolfberry. It also inhibited the growth of *Pseudomonas aeruginosa*. Pseudomonas aeruginosa R5-2 was cultured at 28℃ and 200 rpm for 48 h. The resulting fermentation broth was filtered three times through a 0.22 μm bacterial filter to obtain *Pseudomonas aeruginosa*. Pseudomonas aeruginosa R5-2 fermentation broth supernatant. *Pseudomonas aeruginosa* Pseudomonas aeruginosa R5-2 fermentation broth supernatant was mixed with PDA medium at different volume ratios (2.5%, 5%, 7.5%, 10%, 12.5%) and poured onto plates to ensure the medium was free of Pseudomonas aeruginosa. Pseudomonas aeruginosa PDA plates containing the supernatant of R5-2 fermentation broth were used as a control. Five-mm diameter mycelial cakes were prepared from the edge of activated *Fusarium oxysporum* colonies, inverted and placed in the center of each plate, and incubated at 25°C for 5 days. Colony diameters were measured and inhibition rates were calculated.

[0055] Inhibition rate = (Control colony diameter - Treated colony diameter) / Control colony diameter × 100%

[0056] Pseudomonas aeruginosa Pseudomonas aeruginosa The inhibitory effect of R5-2 fermentation broth supernatant on Fusarium oxysporum is as follows: Figure 4 As shown, Pseudomonas aeruginosa Pseudomonas aeruginosaThe inhibitory effect of the supernatant of R5-2 fermentation broth on Fusarium oxysporum was dose-dependent, with the inhibitory effect increasing with increasing concentration. When Pseudomonas aeruginosa... Pseudomonas aeruginosa When the concentration of R5-2 fermentation broth supernatant was 12.5%, the inhibition rate reached 63.20%, indicating that this *Pseudomonas aeruginosa* strain... Pseudomonas aeruginosa The supernatant of the R5-2 fermentation broth showed significant inhibitory activity against Fusarium oxysporum in the in vitro culture.

[0057] 2. Determination of Pseudomonas aeruginosa Pseudomonas aeruginosa R5-2's antibacterial spectrum

[0058] A well with a diameter of approximately 5 mm was prepared in the center of the PDA medium using a punch, and 20 μL of *Pseudomonas aeruginosa* was inoculated and cultured at 28°C and 200 r / min for 48 h. Pseudomonas aeruginosa The fermentation broth of R5-2 was prepared without the addition of Pseudomonas aeruginosa. Pseudomonas aeruginosa R5-2 fermentation broth was used as a control. Two *Fusarium oxysporum* and *Verticillium dahliae* strains causing root rot of *Lycium barbarum* were selected, along with *Rhizoctonia solani* (root rot of *Potamogeton f. sarcodactylon*), *Verticillium dahliae* (root rot of *Verticillium dahliae*), and *Fusarium graminearum* (root rot of *Fusarium graminearum*) for *Pseudomonas aeruginosa* control. Pseudomonas aeruginosa Antimicrobial spectrum test of R5-2. Five-mm diameter mycelial discs were prepared from the edges of the activated pathogen colonies using a punch. The discs were then inverted on both sides of the hole, with a distance of 4.5cm between them. The discs were incubated at 25℃ until the pathogen colonies on both sides of the control group plate were nearly identical. The width of the inhibition zone in the treatment group was measured, and the inhibition rate was calculated. Inhibition rate = (Inhibition zone width / Distance between discs) × 100%.

[0059] The results are as follows Figure 5 As shown in Table 1, Pseudomonas aeruginosa Pseudomonas aeruginosa R5-2 showed significant antagonistic effects against various pathogenic fungi causing root rot diseases in plants.

[0060] Table 1. Pseudomonas aeruginosa Pseudomonas aeruginosa R5-2's antibacterial rate against different pathogens

[0061]

[0062] Example 3

[0063] This example uses *Pseudomonas aeruginosa*. Pseudomonas aeruginosa Experiment to determine the biocontrol potential of R5-2.

[0064] The experiment used different selective culture media as shown in Table 2 to determine Pseudomonas aeruginosa. Pseudomonas aeruginosa Biocontrol potential of R5-2. First, 20 μL of cultured *Pseudomonas aeruginosa* was inoculated into wells in the center of each plate. Pseudomonas aeruginosaThe R5-2 fermentation broth was incubated at 28°C for 72 hours. Then, *Pseudomonas aeruginosa* was determined using a specific colorimetric reaction or the clear zone method. Pseudomonas aeruginosa R5-2's biological defense potential.

[0065] (1) Iron carrier production capacity: Observe whether an orange transparent ring appears around the colony;

[0066] (2) β-1,3-glucanase production capacity: After staining the plate with 0.2% aniline blue at pH 8.0 for 5 min and decolorizing with sterile water for 1 min, observe whether a clear zone appears around the colony;

[0067] (3) Cellulase production capacity: stain with 0.2% Congo red for 15-20 min, decolorize with 1M NaCl solution for 15 min, fix with 5% acetic acid solution for 10 min, and observe whether a transparent zone appears around the colony.

[0068] (4) Lipase production capacity: Before pouring the plate, add 1.6% neutral red solution to the culture medium at about 60℃, and observe whether a discolored halo is produced after incubation;

[0069] (5) Protease production capacity: Observe whether a clear zone formed by casein decomposition appears around the colony;

[0070] (6) Chitinase production capacity: stain with 0.2% Congo red for 15-20 min, decolorize with 1 mol / L NaCl for 15 min, and observe whether a transparent zone appears around the colony.

[0071] Each experiment was performed in triplicate. The results are as follows: Figure 6 As shown, Pseudomonas aeruginosa Pseudomonas aeruginosa R5-2 has the ability to produce siderophores, β-1,3-glucanase, cellulase, lipase, protease, and chitinase.

[0072] Table 2 Culture medium formulation

[0073]

[0074] Example 4

[0075] This example uses *Pseudomonas aeruginosa*. Pseudomonas aeruginosa Indoor potted plant control test experiment of R5-2 against Fusarium oxysporum. Fusarium oxysporum is the main pathogen of root rot of wolfberry and Fusarium wilt of tomato. The control efficacy was tested by infecting wolfberry and tomato with Fusarium oxysporum.

[0076] 1. Pseudomonas aeruginosa Pseudomonas aeruginosa Efficacy test of R5-2 against Fusarium oxysporum on wolfberry

[0077] First, sterile soil and organic fertilizer were mixed in a 3:1 ratio and then divided into flowerpots. Healthy goji berry seedlings that had grown to about 10cm in height were then transplanted. One week after transplanting, each goji berry plant in the experimental group was drenched with Pseudomonas aeruginosa at the root zone. Pseudomonas aeruginosa R5-2 fermentation broth (OD) 600 =0.8) 10mL, and the control group was treated with an equal volume of sterile water. Treatment was repeated every 2 days for a total of three treatments. Two days after the last treatment, both the experimental and control groups were irrigated with a suspension of Fusarium oxysporum spores (10) 8 (CFU / mL), 10 mL per plant, treated twice, every 2 days. Each group was replicated in triplicate. Disease incidence was observed and photographed on day 42 after pathogen inoculation. Results are as follows: Figure 7 As shown, under indoor potted conditions, wolfberry plants treated with Fusarium oxysporum suffered severe disease, exhibiting symptoms such as leaf drying, wilting, and restricted growth. However, the earlier application of Pseudomonas aeruginosa significantly reduced the incidence of the disease. Pseudomonas aeruginosa The wolfberry plants treated with R5-2 grew normally and showed no obvious symptoms of disease, indicating that *Pseudomonas aeruginosa* was present. Pseudomonas aeruginosa R5-2 has a significant control effect against Fusarium oxysporum on wolfberry.

[0078] 2. Pseudomonas aeruginosa Pseudomonas aeruginosa Efficacy test of R5-2 against Fusarium oxysporum on tomatoes

[0079] The experimental method was the same as above, with tomatoes as the experimental subject. Because tomatoes grow faster than goji berries, the disease incidence was observed and photographed on day 28 after pathogen inoculation. The experimental results are as follows: Figure 8 As shown, under indoor potted conditions, tomato plants treated with Fusarium oxysporum were more severely affected, exhibiting symptoms such as yellowing leaves, wilting, and stunted growth. In contrast, plants treated with the biocontrol agent Pseudomonas aeruginosa showed significantly better results. Pseudomonas aeruginosa Tomato plants treated with R5-2 fermentation broth showed good growth and no obvious disease symptoms, indicating that strain R5-2 has a significant control effect on Fusarium oxysporum in tomatoes.

[0080] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A strain of Pseudomonas aeruginosa, characterized in that, The *Pseudomonas aeruginosa* was named Pseudomonas aeruginosa R5-2, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34796, contains the 16S rDNA sequence of *Pseudomonas aeruginosa* as shown in SEQ ID NO:

1. *Pseudomonas aeruginosa* can produce siderophores, β-1,3-glucanase, cellulase, lipase, protease, and chitinase. It can also inhibit the growth of *Fusarium oxysporum*, *Verticillium dahliae*, *Rhizoctonia solani*, or *Fusarium graminearum*.

2. A microbial agent, characterized in that, The agent contains the Pseudomonas aeruginosa as described in claim 1, wherein the active ingredient of the agent is selected from one or more of the Pseudomonas aeruginosa bacterial suspension and fermentation broth.

3. A method for preparing the microbial agent according to claim 2, characterized in that, This includes culturing the *Pseudomonas aeruginosa* as described in claim 1 and collecting the bacterial cells or fermentation broth of the *Pseudomonas aeruginosa*.

4. The application of the *Pseudomonas aeruginosa* of claim 1 or the bacterial agent of claim 2 in inhibiting the growth of pathogenic bacteria, characterized in that... The pathogens are Fusarium oxysporum, Verticillium dahliae, Rhizoctonia solani, or Fusarium graminearum.

5. The application of *Pseudomonas aeruginosa* as described in claim 1 or the inoculant as described in claim 2 in the prevention and control of root rot in wolfberry, characterized in that, The root rot of wolfberry was induced by Fusarium oxysporum.

6. A method for controlling soil-borne fungal diseases of plants, characterized in that, This includes applying the fungal agent of claim 2 to the rhizosphere of a plant, wherein the plant is wolfberry or tomato, and the soil-borne fungal disease is induced by Fusarium oxysporum.

Images