Pseudomonas chlororaphis S7-24 for antagonizing xanthomonas oryzae and application of pseudomonas chlororaphis S7-24

Abstract

The invention discloses pseudomonas chlororaphis S7-24 for antagonizing xanthomonas oryzae and application of the pseudomonas chlororaphis S7-24, and belongs to the technical field of microorganisms. The pseudomonas chlororaphis S7-24 is preserved in the China Center for Type Culture Collection on November 12, 2025, and the preservation number of the pseudomonas chlororaphis S7-24 is CCTCC NO: M 20252533. The pseudomonas chlororaphis S7-24 provided by the invention is a newly found pseudomonas biocontrol resource, and verification shows that the strain S7-24 has relatively high antagonistic activity on rice bacterial blight and rice bacterial streak, and can be used for effectively preventing and treating rice bacterial blight and rice bacterial streak. The pseudomonas chlororaphis S7-24 provided by the invention is used for preventing and treating rice bacterial leaf blight and rice bacterial leaf streak, can reduce the use amount of chemical pesticides, ensures grain safety, and has better economic and ecological benefits.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Pseudomonas aeruginosa S7-24 antagonistic to Xanthomonas oryzae and its applications. Background Technology

[0002] Rice, a staple food for humankind, has suffered severe yield losses due to bacterial diseases. Among these, Xanthomonas oryzae (…) is a major culprit. Xanthomonas oryzae Two pathogenic species of rice—*Bacterium tumefaciens* (rice bacterial blight) Xanthomonas oryzae pv. Oryzae , Xoo ) and rice bacterial leaf streak ( Xanthomonas oryzae pv. Oryzicola, Xoc Bacterial blight (BB) and bacterial leaf streak (BLS) caused by bacteria can severely damage rice production. They are widespread and frequently occur, usually causing 5%-10% yield loss. In severe outbreaks, the yield reduction can reach more than 50%, and in extreme cases, it can even lead to total crop failure.

[0003] In recent years, with the popularization of simplified rice cultivation, the expansion of two-line hybrid indica rice planting area, and the increase in cross-regional germplasm resource circulation, coupled with the variation in the pathogenicity of the pathogens themselves, the scope of these two bacterial diseases is no longer limited to the main indica rice producing areas in the south, but is rapidly spreading to the indica-japonica mixed planting areas in the Yangtze River Basin and the japonica rice planting areas in the north, posing an increasingly serious threat to rice production safety in many regions. Currently, the mainstream method for controlling rice bacterial blight is still chemical agents, commonly used ones such as thiamethoxam, thiamethoxam, thiamethoxam zinc, and trichloroisocyanuric acid. However, the drawbacks of long-term and large-scale application of chemical pesticides are becoming increasingly prominent: on the one hand, the pathogens' resistance to pesticides continues to increase, causing the control effect of pesticides to decline continuously, forcing growers to increase the dosage; on the other hand, pesticide residues not only affect the food safety of rice, but also cause irreversible damage to the farmland ecosystem. At the same time, the development of new chemical fungicides faces problems such as high cost, long cycle and high risk. In recent years, only a handful of new chemical agents have been approved for registration for the prevention and control of bacterial diseases in crops.

[0004] Biological control, with its advantages of safety, no residue, and good environmental compatibility, has become a key development direction in the global green plant protection field. Microorganisms are one of the core carriers of biological control. Many strains of Bacillus and Pseudomonas have been proven to have good control potential against plant diseases, but the control efficacy varies significantly among different strains, exhibiting strong strain specificity. Currently, regarding *Pseudomonas aeruginosa* (… P. chlororaphisResearch on the application of Pseudomonas aeruginosa in the control of bacterial diseases in rice is still relatively limited. Therefore, screening and developing highly efficient biocontrol strains of Pseudomonas aeruginosa against these diseases is of great practical value in promoting the construction of a green disease control system for rice. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Pseudomonas aeruginosa S7-24 that antagonizes Xanthomonas oryzae and its application, in order to solve the problems existing in the prior art. It has been verified that strain S7-24 has high antagonistic activity against both rice bacterial blight and rice bacterial leaf streak, and can effectively control rice bacterial blight and rice bacterial leaf streak.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a *Pseudomonas aeruginosa* ( Pseudomonas chlororaphis S7-24, the *Pseudomonas aeruginosa* S7-24, was deposited on November 12, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20252533, and the deposit address is Wuhan University, Wuhan, China.

[0007] The present invention also provides a microbial inoculant containing the aforementioned Pseudomonas aeruginosa S7-24 or a suspension thereof.

[0008] This invention also provides the application of the aforementioned *Pseudomonas aeruginosa* S7-24 or the aforementioned microbial agent in inhibiting pathogens, including *Bacillus thuringiensis*, the causal agent of rice bacterial blight. Xanthomonas oryzae pv. Oryzae ) and rice bacterial leaf streak ( Xanthomonas oryzae pv. Oryzicola ).

[0009] The present invention also provides the application of the aforementioned Pseudomonas aeruginosa S7-24 or the aforementioned microbial agent in the prevention and control of plant diseases, including rice bacterial leaf blight and rice bacterial leaf streak.

[0010] The present invention also provides a method for preventing and controlling rice bacterial blight, comprising the step of treating rice leaves with the aforementioned Pseudomonas aeruginosa S7-24 or its bacterial suspension.

[0011] Optionally, the concentration of viable bacteria in the bacterial suspension is 1.0 × 10⁻⁶. 8 cfu / mL.

[0012] The present invention also provides a method for preventing and controlling bacterial leaf streak in rice, comprising the step of treating rice leaves with the aforementioned Pseudomonas aeruginosa S7-24 or its bacterial suspension.

[0013] Optionally, the concentration of viable bacteria in the bacterial suspension is 1.0 × 10⁻⁶. 8cfu / mL.

[0014] The present invention also provides a biocontrol agent for controlling rice bacterial leaf blight and / or rice bacterial leaf streak, the biocontrol agent comprising the aforementioned Pseudomonas aeruginosa S7-24 or a suspension thereof.

[0015] The present invention discloses the following technical effects: The *Pseudomonas aeruginosa* S7-24 provided by this invention is a newly discovered biocontrol resource of *Pseudomonas*. Verification has shown that strain S7-24 exhibits high antagonistic activity against both *Bacillus oryzae* and *Bacillus streak*, effectively controlling these diseases. Using *Pseudomonas aeruginosa* S7-24 provided by this invention for the control of rice bacterial blight and rice bacterial streak can reduce the use of chemical pesticides, ensuring food security and demonstrating significant economic and ecological benefits. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The antagonistic activity of *Pseudomonas aeruginosa* S7-24 against *Bacillus thuringiensis*, the causal agent of rice bacterial blight, was studied. Xoo The fungus causing bacterial blight in rice was used as a control; ddH2O was used as a control. Figure 2 The antagonistic activity of *Pseudomonas aeruginosa* S7-24 against bacterial leaf streak pathogens was studied, among which... Xoc The pathogen is *Bacillus streakus*, the bacterial causal agent of rice leaf streak; ddH2O was used as a control. Figure 3 Statistics on the diameter of the inhibition zone of Pseudomonas aeruginosa S7-24 against strains of rice bacterial blight and bacterial leaf streak; Figure 4 The colony morphology (a) and Gram staining of Pseudomonas aeruginosa S7-24 on LB agar plates (b). Figure 5 Phylogenetic analysis based on the 16S rRNA gene sequence of Pseudomonas aeruginosa S7-24; Figure 6 The inhibitory effect of Pseudomonas aeruginosa S7-24 bacterial suspension on the expansion of bacterial blight lesions in rice; Figure 7 The control effect of Pseudomonas aeruginosa S7-24 bacterial suspension on rice bacterial blight; Figure 8The inhibitory effect of Pseudomonas aeruginosa S7-24 bacterial suspension on the expansion of bacterial leaf streak lesions in rice; Figure 9 The study investigated the control effect of Pseudomonas aeruginosa S7-24 bacterial suspension on bacterial leaf streak in rice. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] The pathogens causing bacterial blight of rice, PXO99 (PXO99A), and the pathogen causing bacterial leaf streak of rice, Rs105, have been published in the literature OsPRMT5 methylates OsPAL1 to promote rice resistance, hindered by aXanthomonas oryzae effector (https: / / doi.org / 10.1111 / jipb.13885).

[0024] LB medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, add water to make up to 1000 mL, pH 7.0±0.2, sterilize at 121℃ for 20 min; solid medium is supplemented with 15 g / L agar powder.

[0025] Example 1: Isolation and Identification of Pseudomonas aeruginosa S7-24 (1) Isolation of bacterial strains to be screened: Several leaf samples from rice fields during the booting stage and infected with bacterial leaf streak were randomly collected. The samples were disinfected with 75% ethanol for 30 seconds, rinsed three times with sterile water, and about 0.3 g of the treated diseased tissue was placed in a sterile 2 mL centrifuge tube. Two sterile steel balls and 1 mL of sterile water were added, the tube was tightly capped, and the tube was shaken vigorously for 2-3 minutes to fully grind and break down the diseased tissue. The tube was allowed to stand at room temperature for 10 minutes, and the supernatant bacterial suspension was collected. The suspension was serially diluted 10-fold with sterile water. 100 μL of each concentration gradient was spread onto LB agar plates and incubated upside down at 28°C for 48 h. Single colonies were picked up with an inoculation loop and streaked twice on LB agar plates for purification. The purified single colonies of different morphologies were transferred to LB agar plates and incubated upside down at 28°C for 48 h. The plates were then stored at 4°C for later use.

[0026] (2) Plate antagonistic screening of biocontrol strains: Each strain purified on LB solid plates was cultured in LB liquid medium at 220 rpm, 28℃, for 16 h, as seed culture for later use; in addition, glycerol strains of rice bacterial blight pathogen PXO99 and rice bacterial leaf streak pathogen Rs105 were streaked onto LB solid plates and cultured at 28℃ for 48 h. Then, single colonies were picked and inoculated into 250 mL Erlenmeyer flasks containing 50 mL of LB liquid medium and cultured at 220 rpm, 28℃ for 20 h. The culture solution (OD) was then compared with the culture medium. 600 ≈1.0) was added to liquid low-temperature LB agar medium at a ratio of 1% (v / v), and quickly mixed to prepare LB solid nutrient plates. 2.5 µL of the seed culture of the test strain was spotted onto the prepared LB solid nutrient plates, with plates without antagonistic bacteria as a control (ddH2O). The treated antagonistic plates were incubated upside down at 28℃ for 48 h. The antagonistic activity of the test strain against *P. xerophyte* PXO99, the causal agent of rice bacterial leaf blight, and *R. xerophyte* Rs105, was assessed by the diameter of the antagonistic zone. Strains with an antagonistic zone diameter greater than 10 mm were then selected. Results are as follows: Figures 1-3As shown, among the 295 strains tested, strain S7-24 performed exceptionally well, with an antagonistic zone diameter of 35.24 mm to 37.88 mm against rice bacterial blight and 13.925 mm to 15.831 mm against rice bacterial leaf streak.

[0027] (3) Morphological observation of biocontrol strain S7-24: Using a sterile inoculation loop, bacterial cells were picked from a plate containing strain S7-24 stored at 4℃ and streaked onto LB agar plates. After culturing at 28℃ for 48 h, colony characteristics were observed: Single colonies were mostly yellowish-green, slightly raised, nearly circular in shape, and 1.5-2 mm in diameter. The colony surface was moist and glossy, with a relatively viscous texture, and the colony edges were neat or slightly wavy. The bacterial cells were Gram-negative, short rod-shaped, and usually arranged singly or in pairs. The peptidoglycan layer in its cell wall structure was relatively thin, and it had an outer membrane structure, which caused it to stain red during Gram staining (…). Figure 4 The colony morphology and Gram staining of S7-24 can correspond to typical Pseudomonas characteristics.

[0028] (4) Molecular identification of biocontrol strain S7-24: Using a single colony on a plate as a template, PCR amplification was performed using the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2) for bacterial 16S rRNA. PCR reaction system (20 μL): 1 μL each of forward and reverse primers, 10 μL of 2×Rapid TagMaster Mix, 1 μL of template DNA, and 7 μL of ddH2O. Reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 90 s, 30 cycles; 72℃ extension for 10 min. The PCR amplification product was taken out, and an appropriate amount (generally 5-10 μL) was spotted into the prepared 1% gel wells for electrophoresis. Electrophoresis was performed at 125 V, with the electrophoresis time varying depending on the size of the target fragment and the length of the electrophoresis tank, typically 30-60 min. The remaining PCR products were sequenced, and the sequencing results were analyzed using BLAST on the NCBI website. Based on the obtained 16S rRNA gene sequence (SEQ ID NO.3) and the GeneBank database, phylogenetic analysis of strain S7-24 was performed using MEGA 11 software. This strain is related to *Pseudomonas aeruginosa* (…). Pseudomonas chlororaphis ) come together ( Figure 5 ).

[0029] SEQ ID NO.3:

[0030] Therefore, strain S7-24 was identified as *Pseudomonas aeruginosa* (…). Pseudomonas chlororaphis The strain, named S7-24, was deposited on November 12, 2025, at the China Center for Type Culture Collection (CCTCCNO: M 20252533) at Wuhan University, Wuhan, China.

[0031] Example 2: Preparation of Pseudomonas aeruginosa strain S7-24 inoculum Preparation of S7-24 biocontrol agent: Glyceryl inoculum of strain S7-24 was streaked onto LB agar plates and incubated at 28°C for 36 h. Single colonies of S7-24 were picked up using a sterile toothpick and inoculated into a 250 mL Erlenmeyer flask containing 50 mL of LB liquid medium. The flask was incubated at 220 rpm and 28°C for 16 h to obtain the seed culture. The seed culture was then inoculated at a ratio of 1% (v / v) into a 1000 mL Erlenmeyer flask containing 200 mL of LB medium and incubated at 220 rpm and 28°C for 16 h to obtain the S7-24 fermentation broth. Conventional adjuvants (2% soluble starch, 0.3% xanthan gum, and 0.1% Tween 80 in this example) were added to the fermentation broth to adjust the pH to 6.5–7.2, resulting in the S7-24 biocontrol agent (1.0 × 10⁻⁶ bacterial concentration). 8 (cfu / mL), then aseptically filled and stored at room temperature.

[0032] Preparation of S7-24 bacterial suspension: The S7-24 seed culture was prepared according to the above method. The S7-24 seed culture was inoculated at a ratio of 1% (v / v) into a 250 mL Erlenmeyer flask containing 50 mL of 50% LB liquid medium. The flask was cultured at 220 rpm and 28℃ for 16 h to obtain the S7-24 fermentation broth. The S7-24 fermentation broth was centrifuged at 5000 rpm for 10 min, the supernatant was removed, and the bacterial cells were collected. The bacterial cells were resuspended in sterile water, washed three times, and the cell density was adjusted to 1.0 × 10⁻⁶. 8 CFU / mL, add Tween 80 to a final concentration of 0.1% (v / v) to prepare S7-24 bacterial suspension.

[0033] Example 3: Control test of Pseudomonas aeruginosa strain S7-24 suspension against rice bacterial blight The rice bacterial blight pathogen strain PXO99, preserved in glycerol from a -80℃ ultra-low temperature freezer, was streaked onto LB agar plates and incubated at 28℃ for 48 h. Single colonies of PXO99 were then picked up with sterile toothpicks and inoculated into 250 mL Erlenmeyer flasks containing 50 mL of LB liquid medium. The flasks were incubated at 220 rpm and 28℃ for 20 h, and the bacterial concentration was adjusted to 1.0 × 10⁻⁶ cells / mL using sterile water.8 The concentration of cfu / mL was increased by adding Tween 80 to a final concentration of 0.1% (v / v) as the pathogen seed culture.

[0034] The rice variety Nipponbare was selected, and three biocontrol treatments were set up: Treatment 1, first sprayed with 1.0×10 8 Treatment 1: Slightly moisten the leaves with a CFU / mL S7-24 bacterial suspension. After 24 hours, cut leaves approximately 3 cm from the leaf tip and inoculate with PXO99 seed solution, labeling the treatment as S7-24+PXO99. Treatment 2: First, cut leaves approximately 3 cm from the leaf tip and inoculate with PXO99 seed solution. After 24 hours, spray with 1.0 × 10⁻⁶ CFU / mL of the solution. 8 A CFU / mL suspension of S7-24 bacteria was used, just enough to slightly moisten the leaf surface, and labeled as PXO99+S7-24; Treatment 3 involved cutting leaves approximately 3 cm from the leaf tip and inoculating them with PXO99 seed solution, labeled as PXO99, as a control treatment. Ten rice plants were inoculated for each treatment, with the top two leaves from each plant inoculated. The length of lesions was assessed 14 days post-inoculation. Results showed that ( Figures 6-7 Compared with the control treatment that was only inoculated with pathogen PXO99, the leaf lesion length of treatment 1 and treatment 2 was significantly reduced (P<0.05), and the difference between the two treatment groups was also significant (P<0.05). The results show that S7-24 can significantly improve the resistance of rice to rice bacterial blight pathogen.

[0035] Example 4: Control test of Pseudomonas aeruginosa strain S7-24 suspension against bacterial leaf streak in rice. The rice bacterial leaf streak pathogen strain Rs105, preserved in glycerol from a -80℃ ultra-low temperature freezer, was streaked onto LB agar plates and incubated at 28℃ for 48 h. A single Rs105 colony was then picked up with a sterile toothpick and inoculated into a 250 mL Erlenmeyer flask containing 50 mL of LB liquid medium. The flask was incubated at 220 rpm and 28℃ for 20 h. The bacterial concentration was then adjusted to 1.0 × 10⁻⁶ cells / mL using sterile water. 8 The concentration of cfu / mL was increased by adding Tween 80 to a final concentration of 0.1% (v / v) as the pathogen seed culture.

[0036] The same rice variety, Nipponbare, was selected, and three biocontrol treatments were set up: Treatment 1, first sprayed with 1.0×10 8 Treatment 1: Slightly moisten the leaves with a CFU / mL S7-24 bacterial suspension. After 24 hours, cut leaves approximately 3 cm from the leaf tip and inoculate with rice bacterial leaf streak seed solution, labeling it as S7-24+Rs105. Treatment 2: First, cut leaves approximately 3 cm from the leaf tip and inoculate with rice bacterial leaf streak Rs105 seed solution. After 24 hours, spray with 1.0×10⁻⁶ CFU / mL of the solution.8 A CFU / mL suspension of S7-24 bacteria was used, just enough to slightly moisten the leaf surface, and labeled as Rs105+S7-24; Treatment 3 involved cutting leaves approximately 3 cm from the leaf tip and inoculating them with seed culture of rice bacterial leaf streak fungus Rs105, labeled as Rs105, as a control treatment. Ten rice plants were inoculated for each treatment, with the top two leaves from each plant inoculated. The length of lesions was assessed 14 days post-inoculation. Results showed that ( Figures 8-9 Compared with the control treatment that was only inoculated with pathogen Rs105, the leaf lesion length of biocontrol treatment 1 and treatment 2 was significantly reduced (P<0.05), and there was no significant difference between the two treatment groups (P>0.05). The results show that S7-24 can significantly improve the resistance of rice to bacterial leaf streak pathogen.

[0037] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A type of Pseudomonas aeruginosa ( Pseudomonas chlororaphis S7-24, characterized in that, The *Pseudomonas aeruginosa* S7-24 was deposited at the China Center for Type Culture Collection (CCTCC) on November 12, 2025, with accession number CCTCC NO: M20252533, and the deposit address is Wuhan University, Wuhan, China.

2. A microbial inoculant, characterized in that, Contains Pseudomonas aeruginosa S7-24 or a suspension thereof as described in claim 1.

3. The application of *Pseudomonas aeruginosa* S7-24 as described in claim 1 or the microbial agent as described in claim 2 in inhibiting pathogenic bacteria, characterized in that... The pathogens include *Bacillus thuringiensis*, the causal agent of rice bacterial blight. Xanthomonas oryzae pv. Oryzae ) and rice bacterial leaf streak ( Xanthomonas oryzae pv. Oryzicola ).

4. The application of *Pseudomonas aeruginosa* S7-24 as described in claim 1 or the microbial agent as described in claim 2 in the prevention and control of plant diseases, characterized in that... The plant diseases mentioned include rice bacterial leaf blight and rice bacterial leaf streak.

5. A method for controlling bacterial leaf blight in rice, characterized in that, The method includes the step of treating rice leaves with Pseudomonas aeruginosa S7-24 or its bacterial suspension as described in claim 1.

6. The method as described in claim 5, characterized in that, The concentration of viable bacteria in the bacterial suspension was 1.0 × 10⁻⁶. 8 cfu / mL.

7. A method for controlling bacterial leaf streak in rice, characterized in that, The method includes the step of treating rice leaves with Pseudomonas aeruginosa S7-24 or its bacterial suspension as described in claim 1.

8. The method as described in claim 7, characterized in that, The concentration of viable bacteria in the bacterial suspension was 1.0 × 10⁻⁶. 8 cfu / mL.

9. A biocontrol agent for controlling rice bacterial leaf blight and / or rice bacterial leaf streak, characterized in that, The biocontrol agent comprises Pseudomonas aeruginosa S7-24 or its bacterial suspension as described in claim 1.

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