Pseudomonas Hunan JP1-10 and application thereof

Abstract

The invention relates to Hunan pseudomonas and application thereof. The pseudomonas hunanensis JP1-10 disclosed by the invention is preserved in the China General Microbiological Culture Collection Center, and the preservation number of the pseudomonas hunanensis JP1-10 is CGMCC (China General Microbiological Culture Collection Center) NO: 33318. The strain disclosed by the invention has relatively high salt resistance, has the functions of producing siderophores, IAA, nitrogen fixation and phosphate solubilization, can help plants relieve salt stress and promote growth, and also can antagonize pathogenic bacteria of fusarium oxysporum of cucumbers. Wide application prospects are realized.

Description

Technical Field

[0001] The invention relates to the field of agricultural microorganisms, and in particular to Hunan Pseudomonas and its application in soil fertilizer. Background Art

[0002] In the field of modern agriculture and environmental science, soil salinization has become an important factor affecting global crop yield and quality. With climate change, improper irrigation management, and the intensification of seawater backflow in coastal areas, soil salt concentration has gradually increased, which not only inhibits plant growth, but may also cause or aggravate the occurrence of plant diseases. In view of this, exploring effective strategies to alleviate the impact of salt stress on plants and enhance the ability of plants to resist pathogens has become a key issue in agricultural science research.

[0003] Pseudomonas, as a type of Gram-negative bacteria, has attracted widespread attention due to its wide ecological distribution and its advantages in promoting plant growth, biological control, environmental protection, etc. These microorganisms can 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, solubilization of nutrients such as phosphorus, and the production of antimicrobial compounds to resist plant pathogens. In particular, certain specific strains of Pseudomonas have been shown to effectively inhibit a variety of important plant pathogenic fungi such as strawberry anthracnose, cucumber wilt, and banana Panama pathogen, providing a powerful tool for agricultural biological control.

[0004] Despite this, the 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, which limits their effectiveness in salinized soils; on the other hand, although some strains show good antibacterial activity, their ability to improve the soil environment and promote plant growth needs to be further improved. Therefore, it is urgent to develop a new Pseudomonas strain that can tolerate salt-alkali stress and achieve multiple functions (for example, promoting plant growth and controlling diseases). Summary of the invention

[0005] The present invention aims to provide a new strain named Hunan Pseudomonas JP1-10 (CGMCC NO: 33318), which is developed under the above background. The strain has high salt tolerance and the characteristics of secreting siderophores, producing IAA, fixing nitrogen and solubilizing phosphorus, and can help plants alleviate salt stress and promote plant growth.

[0006] The Pseudomonas hunanensis JP1-10 strain provided by the present invention is classified and named as Pseudomonas hunanensis, and has been deposited in the General Microbiology Center of the China Culture Collection Administration on January 7, 2025, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC NO: 33318.

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

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

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

[0010] The present invention provides the use of the Hunan Pseudomonas JP1-10 in improving soil quality. The Hunan Pseudomonas JP1-10 of the present invention can produce iron carriers and IAA, has the functions of nitrogen fixation and phosphorus solubilization, and can effectively improve soil quality.

[0011] The present invention also provides the use of the Hunan Pseudomonas JP1-10, fermentation product, fermentation liquid, bacterial suspension or sterile fermentation filtrate or fermentation liquid antibacterial active substance crude extract in preventing and controlling plant diseases. In a specific example, the plant disease is caused by cucumber wilt pathogen.

[0012] In one embodiment, the application of the present invention for preventing and controlling plant diseases is to spray a bacterial solution containing Hunan Pseudomonas JP1-10 on plants; preferably, the bacterial solution containing Hunan Pseudomonas JP1-10 is 10 5 -10 7 cfu / mL.

[0013] The present invention also provides the use of the Hunan Pseudomonas JP1-10, fermentation product, fermentation broth, bacterial suspension or sterile fermentation filtrate or fermentation broth antibacterial active substance crude extract in alleviating salt stress and promoting plant growth. In a specific example, the specific concentration range of the Hunan Pseudomonas JP1-10 used is 10 5 -10 7 cfu / mL, in some specific examples, the growth promotion is specifically promoting the growth of plant roots and aerial parts. In a specific example, the plant is Arabidopsis thaliana or Brassica pekinensis.

[0014] In one embodiment, the application of the present invention in alleviating salt stress and promoting plant growth is specifically to irrigate the bacterial solution containing Hunan Pseudomonas JP1-10 to the roots of plants; preferably, the bacterial solution containing Hunan Pseudomonas JP1-10 is 10 5 -10 7 cfu / mL.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

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

[0017] (2) The Hunan Pseudomonas of the present invention integrates multiple properties that are beneficial to plant health, including but not limited to the ability to produce siderophores, synthesize IAA, fix nitrogen and solubilize phosphate, which helps to improve plant nutrient absorption, reduce the negative effects of salt stress on plants, and promote healthy growth of plants. It has great application value and market development prospects.

[0018] (3) The Hunan Pseudomonas JP1-10 provided by the present invention shows antagonism to the pathogen of cucumber wilt, providing an additional protective barrier for plants and offering 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 the present invention overcome the deficiencies in the prior art, provide an efficient and environmentally friendly solution to soil salinization and plant diseases, and promote the development of sustainable agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the colony morphology of Hunan Pseudomonas JP1-10;

[0022] Figure 2This is a photo of Hunan Pseudomonas JP1-10 after Gram staining;

[0023] Figure 3 This is the result of the iron carrier produced by Hunan Pseudomonas JP1-10, which produces a transparent circle on CAS medium;

[0024] Figure 4 This is the result of the transparent circle produced by Hunan Pseudomonas JP1-10 on nitrogen-free medium;

[0025] Figure 5 This is the result of the transparent circle produced by Hunan Pseudomonas JP1-10 on the PVK plate;

[0026] Figure 6 This is the color development result of IAA production by Hunan Pseudomonas JP1-10;

[0027] Figure 7 This is a photo of the confrontation between Hunan Pseudomonas JP1-10 and cucumber wilt pathogen on a plate;

[0028] Figure 8 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] Fig. 9 Hunan Pseudomonas JP1-10 promoted the growth of root hairs of Arabidopsis thaliana on MS medium with different salt concentrations;

[0030] Fig.10 This is the growth-promoting effect of Hunan Pseudomonas JP1-10 on green vegetables at a salt concentration of 0.4%. DETAILED DESCRIPTION

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

[0032] Unless otherwise specified, the various materials and reagents used in the present invention can be obtained commercially. Similarly, the experimental methods used are conventional methods unless otherwise specified.

[0033] Example 1 Isolation, identification and preparation of bacterial agent of Hunan Pseudomonas JP1-10.

[0034] Hunan Pseudomonas JP1-10 strain was isolated from the rhizosphere soil of Suaeda salsa in saline-alkali land of Lianyungang. Weigh 10g of rhizosphere soil sample and add it into a triangular flask filled with 100ml sterilized distilled water and glass beads, place it in a shaking incubator at 25-30℃ and 100-300rpm for 10-40min, take samples for gradient dilution, and spread them on LB medium (formula: Tryptone: 10g / L, Yeast extract: 5g / L, Sodium chloride (NaCl): 10g / L, pH7.0-7.5.), after culturing for 24-48 hours, select colonies with unique colony morphology and faster growth rate for purification. Through multiple streaking separations, a single strain was obtained, Figure 1 This is a photo of the colonies of JP1-10 streaked on the plate. Figure 2 This is a micrograph of JP1-10 after Gram staining. Subsequently, the strain was subjected to physiological and biochemical identification and 16S rRNA gene sequence analysis to confirm its classification. At the same time, the strain was propagated in large quantities by liquid fermentation and prepared into a bacterial agent for subsequent application research. The results of 16SrDNA sequencing and BLAST comparison showed that the strain had 99% similarity with Hunan Pseudomonas, and it was identified as Hunan Pseudomonas (Pseudomonashunanensis). Its 16SrDNA base sequence is shown in SEQ ID NO.1, which has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on January 7, 2025, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC NO: 33318.

[0035] Pick a single colony of Hunan Pseudomonas JP1-10 and inoculate it into 3-5mL liquid LB medium, and culture it at 28-37℃, 100-300rpm for 16-18h to obtain seed solution. Inoculate the seed solution into a triangular flask filled with 50-300mL liquid LB medium at an inoculation rate of 2-5%, and culture it at 28-37℃, 100-300rpm for 16-24h to obtain Hunan Pseudomonas JP1-10 bacterial solution with a concentration of 10 7 -10 9 cfu / mL. Depending on the amount of bacterial solution used, the culture can be scaled up step by step in this way.

[0036] Example 2 Qualitative detection of growth-promoting function of Hunan Pseudomonas JP1-10

[0037] (1) Siderophore production function test

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

[0039] Hunan Pseudomonas JP1-10 was activated on LB medium, and the activated strain was inoculated into each test medium, cultured at 30-37°C for 1-8 days, and transparent circles were observed around the colonies on the plate, indicating that the strain had the ability to produce iron carriers.

[0040] (2) Phosphate solubilization by bacteria

[0041] (3) PVK medium: Glucose 10g, Ca 3 (PO 4 ) 2 5g, (NH 4 ) 2 SO4 0.5g, NaCl 0.2g, MgSO 4 0.1g, KCL 0.2g, yeast extract 0.5g, MnSO 4 0.002 g, FeSO 4 0.002g, 0.4% bromophenol blue 6mL, distilled water to 1000mL. 121℃, 20min.

[0042] The purified JP1-10 was spotted on a plate containing PVK culture medium and cultured at 30-37°C for 1-8 days. The formation of a transparent zone around the colony was observed, indicating that Hunan Pseudomonas had the ability to solubilize phosphate.

[0043] (3) Nitrogen fixation by bacteria

[0044] Add 10 μL of the activated JP1-10 bacterial suspension to the Axubei nitrogen-fixing medium and culture in a 28°C incubator for 5 days. Observe whether there is a halo or transparent circle on the culture medium to determine the nitrogen-fixing ability of the strain.

[0045] (4) Indoleacetic acid production function test

[0046] Qualitative determination of IAA function of strains: JP1-10 strain was added to TSB medium containing L-tryptophan at a 1-2% inoculum, and incubated at 30-37°C and 170 r·min -1 After shaking for 24-48h, centrifuge at 10000rpm for 5min to obtain the supernatant. Take 100μL of the supernatant and mix it with an equal volume of Salkowski colorimetric solution. Mix 100μL of uninoculated culture medium with an equal volume of Salkowski colorimetric solution as a blank control. React for 30min in a dark environment. If the solution turns red, it means that the strain has IAA ability.

[0047] The results showed that Hunan Pseudomonas JP1-10 could secrete iron carriers and Fe 3+ Bind to form an enzymatic zone around the colony ( Figure 3 ), transparent circles appeared in both nitrogen-free Axubei and PVK medium ( Figure 4 , Figure 5 ), indicating that JP1-10 has the function of nitrogen fixation and phosphorus solubilization. The supernatant of JP1-10 bacterial solution reacts with Salkowski colorimetric solution to produce red ( Figure 6 ), indicating that JP1-10 has the ability to produce IAA.

[0048] Example 3 Inhibitory Effect of Hunan Pseudomonas JP1-10 on Plant Pathogens

[0049] 2. Inhibition of plant pathogenic fungi

[0050] (1) Plant pathogenic fungi tested: Fusarium wilt of cucumber

[0051] (2) Test method:

[0052] The plate confrontation culture method was used: PDA medium (potato 200g, glucose 20g, agar 20g, fixed to 1000mL, pH 6.5-7.5) was used for the solid plate. The pathogen cake (6mm in diameter) was placed in the center of the blank PDA plate, and the JP1-10 strain was inoculated on the corresponding two sides (about 2cm away from the center of the pathogen cake). The PDA plate inoculated with only the pathogen cake was used as the control, and cultured at 28℃ for 5-6 days.

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

[0054] (3) Test results:

[0055] JP1-10 had a strong inhibitory effect on the tested cucumber wilt ( Figure 7 ), the inhibition rate reached more than 70%.

[0056] Example 4 Salt tolerance of Hunan Pseudomonas JP1-10

[0057] The JP1-10 bacterial solution was inoculated into liquid LB with different NaCl concentrations at an inoculum size of 2-5% and cultured at 30-37°C and 150-200rpm for 3-7 days. The results showed that JP1-10 could grow at a NaCl concentration of 18% (see Table 2) and had high salt tolerance.

[0058] Table 2 Salt tolerance of Hunan Pseudomonas JP1-10

[0059]

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

[0061] In summary, Hunan Pseudomonas JP1-10 has broad salt tolerance and can still function in extreme environments.

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

[0063] (1) Experimental methods:

[0064] Seed disinfection: Soak Arabidopsis seeds in deionized water for 10 minutes. Remove the unfilled seeds floating on the water surface. Disinfect the seeds with 10% NaClO and shake for 15 minutes. Wash the seeds with sterile water for 5 times and place the washed seeds at 4°C for 3 days.

[0065] Prepare MS culture medium plates containing 0mM, 20mM, 40mM, 60mM, and 80mM NaCl, and dot sterilized Arabidopsis seeds every 1cm on MS plates with different gradient salt concentrations, with 7 seeds per plate. Seal the culture dish with sealing film and place it vertically in a light incubator at 22-25℃ for 7 days, with a light cycle of 16h light and 8h dark. After 7 days, inoculate JP1-10 at the bottom of the plate, and use the uninoculated plate as a control. Put it in the incubator for 7 days, and observe the growth and root system of Arabidopsis after 14 days.

[0066] (2) Experimental results

[0067] Figure 8 Comparison of the growth of Arabidopsis thaliana under different salt concentrations with and without JP1-10 inoculation. Fig. 9 The results show that the root system of Arabidopsis thaliana inoculated with JP1-10 strains is more developed than that of the control under different salt stresses, the number of root hairs is significantly increased, and the growth of Arabidopsis thaliana is significantly better than that without JP1-10 inoculation.

[0068] Example 6 Hunan Pseudomonas JP1-10 alleviates salt stress and promotes growth of green vegetables.

[0069] Test materials: The Chinese cabbage variety used in the experiment was “Heat-Resistant 605”.

[0070] Experimental design: The study was conducted in a greenhouse at the Nanjing Institute of Agricultural Sciences in the hilly area of ​​Jiangsu Province using a potted plant experiment. The flowerpots were 178mm×190mm in size, and each pot was filled with 2.5kg of soil. 10g of NaCl was dissolved in 800mL of deionized water and poured into the pot. The exudate was then poured back to ensure that the NaCl solution was evenly infiltrated into the soil. The soil salt content was 0.4%. Select pakchoy seeds with full grains, sow 20 seeds per pot, and thin them out when they grow to 2 leaves and 1 heart, leaving 5 neat and good pakchoy seedlings in each pot. On the 10th day, inoculate with a heavy suspension of JP1-10 bacteria that had been fermented and cultured for 36 hours to ensure that the inoculation amount was 10 per gram of soil. 6 Each treatment was repeated for 5 pots, with the uninoculated pots as the control. Growth and physiological indexes were measured after 1 month, and the results are shown in Table 3 and Fig.10 shown.

[0071] From Table 3 and Fig.10 It can be seen that inoculation with JP1-10 can effectively help green vegetables alleviate salt stress and promote their growth. Plant height, root length, aboveground weight, underground weight, leaf area, and SPAD value all showed an increasing trend, among which aboveground weight, root length, and maximum leaf area increased significantly.

[0072] Table 3 Growth-promoting effect of Hunan Pseudomonas JP1-10 on green vegetables at a salt concentration of 0.4%

[0073]

Claims

1. A Hunan Pseudomonas JP1-10, classified and named as Hunan Pseudomonas (Pseudomonas hunanensis), and the strain preservation number is CGMCC NO:33318.

2. A fermentation product comprising the Hunan Pseudomonas JP1-10 according to claim 1; preferably, the fermentation product is a fermentation broth containing the strain, a sterile filtrate of the fermentation broth, or a crude extract of antibacterial active substances in the fermentation broth.

3. A bacterial suspension comprising the Hunan Pseudomonas JP1-10 according to claim 1.

4. Use of the Hunan Pseudomonas JP1-10 described in claim 1 in improving soil quality.

5. Use of the Hunan Pseudomonas JP1-10 described in claim 1 or the fermentation product described in claim 2 or the bacterial suspension described in claim 3 in plant disease prevention and control; preferably, the plant disease is a disease caused by cucumber wilt pathogen.

6. The use according to claim 5, characterized in that: Specifically, the bacterial solution containing Hunan Pseudomonas JP1-10 is sprayed on the plants; preferably, the bacterial solution containing Hunan Pseudomonas JP1-10 is 10 5 -10 7 cfu / mL.

7. Use of the Hunan Pseudomonas JP1-10 according to claim 1, the fermentation product according to claim 2, or the bacterial suspension according to claim 3 in alleviating salt stress and promoting plant growth.

8. The use according to claim 7, characterized in that: The concentration range of Hunan Pseudomonas JP1-10 is 10 5 -10 7 cfu / mL.

9. The use according to claim 7, characterized in that: Specifically, the bacterial solution containing Hunan Pseudomonas JP1-10 is irrigated to the roots of plants.

10. The use according to claim 7, characterized in that: The plant is Arabidopsis thaliana or Brassica rapa.

Images