Pseudomonas huanghuannensis JP1-10 and application thereof

Abstract

The present application relates to a kind of Pseudomonas hunanensis and its application.The Pseudomonas hunanensis (Pseudomonas hunanensis) JP1-10 described in the present application has been preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO:33318.The strain of the present application has high salt tolerance, has the functions of producing iron carrier, IAA, nitrogen fixation and phosphorus solubilization, can help plants to relieve salt stress and promote growth, and can also antagonize cucumber fusarium wilt pathogen.The application prospect is wide.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology, and more particularly to a type of Pseudomonas hunanense and its application in soil fertilization. Background Technology

[0002] In the fields of modern agriculture and environmental science, soil salinization has become a significant factor affecting global crop yield and quality. With climate change, inappropriate irrigation management, and the increasing prevalence of seawater intrusion in coastal areas, soil salinity is gradually rising. This not only inhibits plant growth but may also trigger or exacerbate plant diseases. Therefore, exploring effective strategies to mitigate the effects of salt stress on plants and enhance their resistance to pathogens has become a key issue in agricultural scientific research.

[0003] Pseudomonas, a genus of Gram-negative bacteria, has attracted widespread attention due to its broad ecological distribution and its advantages in promoting plant growth, biocontrol, and environmental protection. These microorganisms promote plant health through various mechanisms, including but not limited to the synthesis of plant hormones (such as indole-3-acetic acid, IAA), nitrogen fixation, the dissolution of nutrients such as phosphorus, and the production of antimicrobial compounds to resist plant pathogens. In particular, certain specific Pseudomonas strains have been shown to effectively inhibit several important plant pathogenic fungi, such as *Amanita muscaria* (strawberry anthracnose), *Fusarium wilt* (cucumber wilt), and *Panama disease* (banana wilt), providing a powerful tool for agricultural biocontrol.

[0004] Nevertheless, currently used Pseudomonas strains still face some challenges in actual agricultural production. On the one hand, some strains have limited adaptability to saline-alkali environments, limiting their effectiveness in saline soils; on the other hand, although some strains exhibit good antibacterial activity, their ability to improve soil conditions and promote plant growth needs further improvement. Therefore, developing a novel Pseudomonas strain that can tolerate saline-alkali stress and achieve multiple functions (e.g., promoting plant growth and controlling diseases) is an urgent need. Summary of the Invention

[0005] The purpose of this invention is to provide a novel strain named *Pseudomonas hunanense* JP1-10 (CGMCC NO:33318), which was developed under the aforementioned background. This strain exhibits high salt tolerance, secretion of siderophores, production of IAA, nitrogen fixation, and phosphorus solubilization, thus helping plants alleviate salt stress and promote plant growth.

[0006] The *Pseudomonas hunanensis* strain JP1-10 provided by this invention, classified and named *Pseudomonas hunanensis*, was deposited on January 7, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC NO:33318.

[0007] The present invention also provides a fermentation product prepared from the *Pseudomonas hunanensis* JP1-10 described in the present invention. The fermentation product described in the present invention can be any form of fermented product conventional in the art, such as including but not limited to fermentation broth containing the strain, sterile filtrate of fermentation broth, crude extract of antibacterial active substances from fermentation broth, etc.

[0008] The present invention also provides a bacterial suspension containing the aforementioned Pseudomonas hunanensis JP1-10, which can be a solution obtained by resuspending the centrifuged bacterial cells according to conventional methods and reagents in the art.

[0009] The present invention also provides a crude extract of antibacterial active substances from the fermentation broth of Pseudomonas hunanensis JP1-10.

[0010] This invention provides the application of the Hunan Pseudomonas JP1-10 in improving soil quality. The Hunan Pseudomonas JP1-10 of this invention can produce siderophores and IAA, and has the functions of nitrogen fixation and phosphorus solubilization, which can effectively improve soil quality.

[0011] This invention also provides the application of the aforementioned *Pseudomonas hunanensis* JP1-10, fermentation products, fermentation broth, bacterial suspension, or sterile fermentation filtrate or crude extract of antibacterial active substances in the control of plant diseases. In a specific example, the plant disease is a disease caused by *Fusarium wilt*.

[0012] In one embodiment, the application of the present invention for controlling plant diseases specifically involves spraying a bacterial solution containing *Pseudomonas hunanense* JP1-10 onto the plants; preferably, the bacterial solution containing *Pseudomonas hunanense* JP1-10 is 10... 5 -10 7 cfu / mL.

[0013] This invention also provides the application of *Pseudomonas hunanensis* JP1-10, fermentation products, fermentation broth, bacterial suspension, or sterile fermentation filtrate or crude extract of antibacterial active substances in alleviating salt stress and promoting plant growth. In a specific example, the specific concentration range of *Pseudomonas hunanensis* JP1-10 used is 10. 5 -10 7 In some specific instances, the growth promotion refers to promoting the growth of both the plant roots and above-ground parts. In one specific instance, the plant is Arabidopsis thaliana or Chinese cabbage.

[0014] In one embodiment, the application of the present invention in alleviating salt stress and promoting plant growth specifically involves irrigating the plant roots with a bacterial solution containing *Pseudomonas hunanense* JP1-10; preferably, the bacterial solution containing *Pseudomonas hunanense* JP1-10 has a concentration of 10... 5 -10 7 cfu / mL.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] (1) The Hunan Pseudomonas JP1-10 described in this invention can tolerate 18% NaCl concentration, exhibiting significant salt tolerance characteristics and can withstand high concentrations of salt without losing its physiological activity.

[0017] (2) The Hunan Pseudomonas of the present invention integrates a number of characteristics that are beneficial to plant health, including but not limited to siderophore generation, IAA synthesis, nitrogen fixation and phosphorus solubilization, which helps to improve plant nutrient absorption, reduce the negative impact of salt stress on plants, and promote healthy plant growth. It has great application value and market development prospects.

[0018] (3) The Hunan Pseudomonas JP1-10 provided by the present invention shows antagonistic effect against the pathogen of cucumber wilt, providing an additional protective barrier for plants and providing a new option for disease management in agricultural practice.

[0019] (4) The Hunan Pseudomonas JP1-10 provided by the present invention can effectively alleviate salt stress and promote the growth of plant roots and aboveground parts.

[0020] In summary, the Hunan Pseudomonas JP1-10 strain and its application provided by this invention overcome the shortcomings of the prior art, providing an efficient and environmentally friendly solution for addressing soil salinization and plant diseases, and promoting the development of sustainable agriculture. Attached Figure Description

[0021] Figure 1 This is a colony morphology diagram of Pseudomonas hunanense JP1-10.

[0022] Figure 2Photographs of Hunan Pseudomonas JP1-10 cells after Gram staining;

[0023] Figure 3 Image showing the results of siderophore production by Pseudomonas hunanense JP1-10 on CAS medium, producing clear zones.

[0024] Figure 4 This image shows the results of clear zones produced by *Pseudomonas hunanense* JP1-10 on nitrogen-free medium.

[0025] Figure 5 This image shows the results of Hunan Pseudomonas JP1-10 producing a clear zone on a PVK plate.

[0026] Figure 6 Image showing the chromogenic results of IAA production by Pseudomonas hunanensis JP1-10;

[0027] Figure 7 Photograph of Hunan Pseudomonas JP1-10 and cucumber wilt pathogen confronting each other on a plate;

[0028] Figure 8 To investigate the effect of Hunan Pseudomonas JP1-10 on alleviating salt stress and promoting growth of Arabidopsis thaliana on MS medium with different salt concentrations;

[0029] Figure 9 To promote root hair growth in Arabidopsis thaliana on MS medium with different salt concentrations using Pseudomonas hunanense JP1-10;

[0030] Figure 10 The effect of Hunan Pseudomonas JP1-10 on the growth-promoting effect of 0.4% salt concentration on Chinese cabbage. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and embodiments, but the present invention is by no means limited to these embodiments. The following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0032] Unless otherwise specified, all materials and reagents used in this invention are commercially available. Similarly, unless otherwise specified, the experimental methods used are conventional methods.

[0033] Example 1: Isolation, identification and preparation of fungal agent of Pseudomonas hunanensis JP1-10.

[0034] The *Pseudomonas hunanensis* strain JP1-10 was isolated from the rhizosphere soil of *Suaeda salsa* in Lianyungang, a saline-alkali land. 10g of rhizosphere soil sample was weighed and added to a 100ml Erlenmeyer flask containing sterile distilled water and glass beads. The flask was incubated at 25-30℃ and 100-300rpm for 10-40 minutes. After serial dilution, the sample was plated on LB agar (formulation: tryptone: 10g / L, yeast extract: 5g / L, sodium chloride (NaCl): 10g / L, pH 7.0-7.5). After 24-48 hours of incubation, colonies with unique morphology and rapid growth were selected for purification. A single strain was obtained through repeated streak plating. Figure 1 Photographs of bacterial colonies from JP1-10 streaks. Figure 2 The image shows a Gram-stained micrograph of JP1-10. Subsequently, physiological and biochemical identification and 16S rRNA gene sequence analysis were performed on the strain to confirm its classification. Simultaneously, the strain was extensively propagated using liquid fermentation to prepare a bacterial agent for subsequent research applications. 16S rDNA sequencing and BLAST alignment results showed that this strain had 99% similarity to *Pseudomonas shunanensis*, thus identifying it as *Pseudomonas shunanensis*. Its 16S rDNA base sequence is shown in SEQ ID NO.1 and was deposited on January 7, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO: 33318.

[0035] Single colonies of *Pseudomonas hunanense* JP1-10 were picked and inoculated into 3-5 mL of liquid LB medium. The culture was incubated at 28-37℃ with shaking at 100-300 rpm for 16-18 hours to obtain a seed culture. The seed culture was then inoculated into Erlenmeyer flasks containing 50-300 mL of liquid LB medium at an inoculation rate of 2-5%. The culture was incubated at 28-37℃ with shaking at 100-300 rpm for 16-24 hours to obtain a *Pseudomonas hunanense* JP1-10 bacterial suspension with a concentration of 10%. 7 -10 9 cfu / mL. The culture volume can be scaled up stepwise according to the amount of bacterial culture used.

[0036] Example 2 Qualitative Detection of the Growth-Promoting Function of *Pseudomonas aeruginosa* JP1-10 in Hunan

[0037] (1) Functional testing of iron-producing carrier

[0038] Test culture medium: Ferrophilic detection medium: CAS 0.7g, FeCl3 solution (containing 10mM HCl) 100mL, HDTMA 0.6g, agar 20g, adjusted to 1000mL, pH 7.0.

[0039] Hunan Pseudomonas JP1-10 was activated on LB medium. The activated strain was then inoculated into various test media and cultured at 30-37℃ for 1-8 days. If a clear zone appeared around the colony on the plate, it indicated that the strain had the ability to produce siderophores.

[0040] (2) Phosphate-solubilizing activity of bacteria

[0041] (3) PVK medium: 10g glucose, 5g Ca3(PO4)2, 0.5g (NH4)2SO4, 0.2g NaCl, 0.1g MgSO4, 0.2g KCl, 0.5g yeast extract, 0.002g MnSO4, 0.002g FeSO4, 6mL 0.4% bromophenol blue, and distilled water to a final volume of 1000mL. Incubate at 121℃ for 20min.

[0042] The purified JP1-10 bacteria were seeded on plates containing PVK medium and incubated at 30-37℃ for 1-8 days. The presence of a clear zone around the colony was observed, indicating that *Pseudomonas hunanense* has phosphate-solubilizing activity.

[0043] (3) Nitrogen fixation by bacteria

[0044] Add 10 μL of the activated JP1-10 bacterial suspension to Ashube nitrogen fixation medium and incubate at 28°C for 5 days. Observe whether there is a halo or a clear zone on the medium to determine the nitrogen fixation function of the strain.

[0045] (4) Functional detection of indoleacetic acid production

[0046] Qualitative determination of IAA function of strain JP1-10: Add strain JP1-10 to TSB medium containing L-tryptophan at an inoculum of 1-2%, incubate at 30-37℃ and 170 rpm. -1 After shaking for 24-48 hours, centrifuge at 10,000 rpm for 5 minutes to obtain the supernatant. Take 100 μL of the supernatant and mix it with an equal volume of Salkowski colorimetric solution. Use 100 μL of uninoculated culture medium mixed with an equal volume of Salkowski colorimetric solution as a blank control. React under dark conditions for 30 minutes. If the solution turns red, it indicates that the strain has the ability to produce IAA.

[0047] The results showed that *Pseudomonas hunanense* JP1-10 can secrete siderophores, which interact with Fe in the detection plate. 3+ Enzymatic hydrolysis zones are formed around the colony. Figure 3 Clear zones appeared in both nitrogen-free Assumption and PVK media. Figure 4 , Figure 5This indicates that JP1-10 has nitrogen-fixing and phosphorus-solubilizing effects. The supernatant of the JP1-10 bacterial culture reacts with Salkowski colorimetric solution to produce a red color ( ). Figure 6 This indicates that JP1-10 has the ability to produce IAA.

[0048] Example 3: Inhibitory effect of *Pseudomonas hunanense* JP1-10 on plant pathogens.

[0049] 2. Inhibitory effect on plant pathogenic fungi

[0050] (1) Pathogenic fungi of tested plants: Fusarium wilt of cucumber

[0051] (2) Experimental methods:

[0052] The plate confrontation culture method was used: solid plates were prepared with PDA medium (200g potato, 20g glucose, 20g agar, adjusted to 1000mL, pH 6.5-7.5). A pathogenic fungal disc (6mm in diameter) was placed in the center of a blank PDA plate, and JP1-10 strain was inoculated on both sides (approximately 2cm from the center of the pathogenic fungal disc). PDA plates inoculated only with pathogenic fungal discs served as controls. The plates were incubated at 28℃ for 5-6 days.

[0053] Pathogen inhibition rate = [(pathogen growth diameter in control group - pathogen growth diameter in treatment group) / pathogen growth diameter in control group] × 100%.

[0054] (3) Experimental results:

[0055] JP1-10 showed strong inhibitory effects on all tested cucumber wilt diseases. Figure 7 The inhibition rate reached over 70%.

[0056] Example 4: Salt tolerance of *Pseudomonas hunanense* JP1-10

[0057] JP1-10 bacterial culture was inoculated into liquid LB with different NaCl concentrations at an inoculation rate of 2-5%, and cultured at 30-37℃ and 150-200rpm for 3-7 days. The results showed that JP1-10 could grow under the condition of NaCl mass concentration of 18% (see Table 1) and had the ability to tolerate high salt.

[0058] Table 1 Salt tolerance of *Pseudomonas hunanensis* JP1-10

[0059]

[0060] Note: "+" in the table indicates bacterial growth.

[0061] In summary, Hunan Pseudomonas JP1-10 exhibits broad salt tolerance and can still function effectively under extreme conditions.

[0062] Example 5: Growth-promoting effect of Hunan Pseudomonas JP1-10 in alleviating salt stress in Arabidopsis thaliana.

[0063] (1) Experimental method:

[0064] Seed disinfection: Soak Arabidopsis seeds in deionized water for 10 minutes. Remove any unripe seeds floating on the surface. Disinfect the seeds with 10% NaClO, shaking for 15 minutes. Rinse the seeds five times with sterile water, and then store the rinsed seeds at 4°C for 3 days.

[0065] Prepare MS medium plates containing 0mM, 20mM, 40mM, 60mM, and 80mM NaCl. Place sterilized Arabidopsis seeds every 1cm on each MS plate with different salt concentrations, using 7 seeds per plate. Seal the plates with film and incubate them vertically in a light incubator at 22-25℃ for 7 days (16h light, 8h dark). After 7 days, streak JP1-10 onto the bottom of the plates, using uninoculated plates as a control. Incubate again for 7 days. Observe the growth and root development of Arabidopsis after 14 days.

[0066] (2) Experimental Results

[0067] Figure 8 To compare the growth of Arabidopsis thaliana under different salt concentrations with and without JP1-10 inoculation. Figure 9 The study compared the number of root hairs in Arabidopsis thaliana treated with JP1-10 with that treated without JP1-10. The results showed that under different salt stresses, the root system of Arabidopsis thaliana inoculated with JP1-10 was more developed than that of the control, the number of root hairs was significantly increased, and the growth of Arabidopsis thaliana was also significantly better than that of the uninoculated treatment.

[0068] Example 6: Effects of Hunan Pseudomonas JP1-10 on alleviating salt stress and promoting growth in Chinese cabbage.

[0069] Test materials: The Chinese cabbage variety used in the experiment was "Heat-resistant 605".

[0070] Experimental Design: This study employed pot experiments conducted in a greenhouse at the Nanjing Agricultural Science Research Institute in the hilly region of Jiangsu Province. The pots were 178mm × 190mm in size, each containing 2.5kg of soil. 10g of NaCl was dissolved in 800mL of deionized water and poured into the pots. The exudate was then used to water the soil again, ensuring the NaCl solution was evenly distributed throughout the soil, resulting in a soil salinity of 0.4%. Plump Chinese cabbage seeds were selected, with 20 seeds sown per pot. Once the seedlings reached two leaves and one bud, they were thinned, leaving five uniform and healthy seedlings per pot. On the 10th day, a resuspension of JP1-10 bacteria, fermented for 36 hours, was inoculated, ensuring an inoculum density of 10 g of soil per gram. 6CFU. Each treatment was repeated in 5 pots, with uninoculated pots serving as the control. Growth and physiological parameters were measured after 1 month, and the results are shown in Table 2. Figure 10 As shown.

[0071] From Table 2 and Figure 10 It can be seen that inoculation with JP1-10 can effectively help alleviate salt stress in Chinese cabbage and promote its growth. Plant height, root length, aboveground weight, underground weight, leaf area, and SPAD value all showed an increasing trend, with aboveground weight, root length, and maximum leaf area showing significant increases.

[0072] Table 2. Growth-promoting effect of *Pseudomonas hunanensis* JP1-10 on leafy greens at a salt concentration of 0.4%.

[0073]

Claims

1. A strain of *Pseudomonas hunanense* JP1-10, classified and named *Pseudomonas hunanense* ( Pseudomonas hunanensis The strain has the preservation number CGMCC NO:33318.

2. A bacterial suspension containing the Hunan Pseudomonas JP1-10 as described in claim 1.

3. The application of Hunan Pseudomonas JP1-10 as described in claim 1 in improving soil quality.

4. The application of Hunan Pseudomonas JP1-10 as described in claim 1 in the control of diseases caused by Cucumber Fusarium wilt.

5. The application according to claim 4, characterized in that, Specifically, the bacterial solution containing Hunan Pseudomonas JP1-10 was sprayed onto the plants.

6. The application according to claim 5, characterized in that, The bacterial suspension containing *Pseudomonas hunanense* JP1-10 was 10. 5 -10 7 cfu / mL.

7. The application of Hunan Pseudomonas JP1-10 as described in claim 1 in alleviating salt stress and promoting the growth of Arabidopsis thaliana or Chinese cabbage.

8. The application according to claim 7, characterized in that, The recommended concentration range for Hunan Pseudomonas JP1-10 is 10. 5 -10 7 cfu / mL.

9. The application according to claim 8, characterized in that, Specifically, the bacterial solution containing Hunan Pseudomonas JP1-10 is applied to the roots of Arabidopsis thaliana or Chinese cabbage.

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