A salt-tolerant Pseudomonas alcaligenes strain and its application in improving plant salt stress resistance

Abstract

The present invention discloses a salt-tolerant Pseudomonas alcaligenes strain and its use in improving plant salt stress resistance, belonging to the field of microbial technology. This Pseudomonas alcaligenes strain is isolated from saline-alkali soil and has strong salt tolerance, with a salinity tolerance range of 0-90 g / L. It can also improve plant salt stress resistance and promote plant growth. The strain is deposited with the General Microbiology Center of the China National Center for Microbiological Culture Collection, Beijing, China, on May 19, 2025, under the deposit number CGMCC No. 34593.

Description

Technical Field

[0001] The present invention relates to a bacterium and an application thereof, in particular to a salt-tolerant Pseudomonas alcaligenes strain and an application thereof in improving the salt stress resistance of plants, belonging to the technical field of microorganisms. Background Art

[0002] Food crops are subject to a variety of abiotic stresses during their growth process, and saline-alkali stress is an important one among them. It will affect protein synthesis, lipid metabolism, etc. by limiting the photosynthetic activity of plants, thereby hindering plant growth and reducing yield.

[0003] To increase the usability of salinized soils and improve crop yields, researchers have developed various technologies and approaches to address soil salinization, including physical methods relying on water conservancy projects, chemical methods using chemical reagents, and biological methods relying on various life activities. Among these, plant growth-promoting rhizosphere bacteria (PGPR) have been widely studied and applied in salinized soil improvement and remediation due to their ability to promote plant growth, improve saline-alkali soils, and enhance plant salt stress tolerance, while also being low-cost, environmentally friendly, and pollution-free. PGPRs that enhance plant salt stress tolerance and improve salinized soils are also known as ST-PGPRs (Salt Tolerance-related PGPRs). ST-PGPRs enhance plant salt stress tolerance through various means, including enhancing nutrient availability, improving the soil microenvironment, and altering plant gene expression. For example, ST-PGPR directly synthesizes or regulates the synthesis of plant auxins such as indoleacetic acid, gibberellins, and cytokinins, promoting plant growth and enhancing plant resistance to salt stress. ST-PGPR secretes 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which cleaves ACC into ammonia and α-ketobutyrate. The cleavage products are consumed by bacteria as nitrogen and carbon sources. ACC deaminase not only improves plant survival in saline soils, but also increases plant productivity. ST-PGPR can accumulate osmotic protectants such as proline, glycine, betaine, polyamines, quaternary ammonium compounds, and other amino acids for plants to cope with salt stress. ST-PGPR secretes extracellular polysaccharides, providing a physical barrier for plant roots and supporting plant growth under high salt stress. ST-PGPR stimulates plants to produce antioxidant enzymes to alleviate the negative effects of abiotic stress. ST-PGPR increases mineral absorption and protects plants from ion toxicity.

[0004] Salt-tolerant PGRPs with plant growth-promoting properties are important microbial resources. Current research on PGRPs focuses on Bacillus species, with relatively little research on other genera with salt-tolerant and plant growth-promoting properties. Furthermore, most PGRPs cannot tolerate saline-alkali environments and are unable to grow and reproduce in saline-alkali soils, limiting their application in saline-alkali environments. Given the urgent need for plant cultivation and soil improvement in saline-alkali soils, there is an urgent need to supplement the resources of bacterial strains that are salt-tolerant, promote plant growth, and enhance plant salt-alkali tolerance. Summary of the Invention

[0005] The purpose of the present invention is to provide a salt-tolerant Pseudomonas alcaligenes strain and application thereof in improving plant salt stress resistance.

[0006] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0007] A strain of Pseudomonas pseudoalcaligenes HY-B is provided. The strain is isolated from saline-alkali soil, has strong salt tolerance, a salinity tolerance range of 0-90 g / L, and can enhance plant salt stress resistance and promote plant growth. The strain is deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is May 19, 2025, the deposit number is CGMCC No. 34593, and the classification name is Pseudomonas pseudoalcaligenes.

[0008] The use of the aforementioned Pseudomonas pseudoalcaligenes HY-B in improving plant salt stress resistance; preferably, the plant is corn.

[0009] The benefits of the present invention are that the screened pseudoalcaligenes HY-B has strong salt tolerance (salinity tolerance range is 0-90g / L), can enhance plant salt stress resistance, and promote plant growth, and is a plant growth-promoting bacterium. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is the colony morphology of strain HY-B on LB solid medium;

[0011] Figure 2 This is the calculation result of the amount of IAA produced by strain HY-B;

[0012] Figure 3 This is a graph showing the solubility of inorganic phosphorus in strain HY-B;

[0013] Figure 4 This is a diagram of the siderophore production of strain HY-B;

[0014] Figure 5This is a graph showing the effect of strain HY-B on the growth of corn seedlings in soils with different degrees of salinization;

[0015] Figure 6 This is a graph showing the measurement results of the wet weight of corn seedlings in soils with different degrees of salinization;

[0016] Figure 7 This is a graph showing the measurement results of corn seedling height in soils with different degrees of salinization;

[0017] Figure 8 This is a graph showing the measurement results of the root length of corn seedlings in soils with different degrees of salinization. DETAILED DESCRIPTION

[0018] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0019] 1. Prepare culture medium

[0020] 1. High salt inorganic phosphorus liquid culture medium

[0021] The formula for high-salt, inorganic phosphate liquid culture medium is as follows: NaCl, 50 g; glucose, 10 g; (NH₄)₂SO₄, 0.5 g; yeast extract, 0.5 g; KCl, 0.3 g; MgSO₄, 0.3 g; FeSO₄, 0.03 g; MnSO₄, 0.03 g; Ca₃(PO₄)₂, 5.0 g; and ultrapure water, 1 L. Sterilize and set aside.

[0022] 2. High salt inorganic phosphorus solid culture medium

[0023] The formula of the high-salt inorganic phosphorus solid medium is as follows: add 15g of agar to the above high-salt inorganic phosphorus liquid medium formula. Sterilize and set aside.

[0024] 3. Salt-free (NaCl) inorganic phosphorus solid culture medium

[0025] The formula for salt (NaCl)-free, inorganic phosphate solid medium is as follows: glucose, 10 g; (NH₄)₂SO₄, 0.5 g; yeast extract, 0.5 g; KCl, 0.3 g; MgSO₄, 0.3 g; FeSO₄, 0.03 g; MnSO₄, 0.03 g; Ca₃(PO₄)₂, 5.0 g; agar, 15 g; ultrapure water, 1 L. Sterilize and set aside.

[0026] 4. LB liquid medium

[0027] The formula of LB liquid medium is as follows: tryptone, 10 g; yeast extract, 5 g; NaCl, 10 g; ultrapure water, 1 L. Sterilize and set aside.

[0028] 5. LB solid medium

[0029] The formula for LB solid medium is as follows: add 15 g of agar to the above LB liquid medium formula. Sterilize and set aside.

[0030] 6. CAS detection plate

[0031] The CAS assay plate formulation is as follows: chrome azurol S, 60.5 mg; hexadecyltrimethylammonium bromide, 72.9 mg; ferric chloride hexahydrate, 2.645 mg; sodium dihydrogen phosphate dihydrate, 295.25 mg; disodium hydrogen phosphate dodecahydrate, 1213.5 mg; ammonium chloride, 125 mg; potassium dihydrogen phosphate, 37.5 mg; sodium chloride, 62.5 mg; agar, 9000 mg; ultrapure water, 1 L; pH 6.8 ± 0.1. Sterilize and use.

[0032] 2. Screening and identification of salt-tolerant Pseudomonas alcaligenes

[0033] Soil samples were collected from saline-alkali land in Lijin County, Dongying City, Shandong Province (37.787798°N, 118.41895136°E).

[0034] 2 g of soil sample was added to high-salt inorganic phosphorus liquid culture medium and cultured in a constant temperature shaker at 30°C and 160 rpm for 5 days. Then 1 mL of bacterial solution was aspirated, diluted and evenly spread on high-salt inorganic phosphorus solid culture medium, and cultured in a constant temperature incubator at 30°C for 48 hours. After that, single colonies that produced phosphate-dissolving circles on the plate were selected, streaked on LB solid culture medium using the three-line method, and cultured in a constant temperature incubator at 30°C for 12 hours to obtain the strain, which was recorded as HY-B.

[0035] The colony morphology of strain HY-B on LB solid medium is as follows Figure 1 As shown, it appears as light yellow, irregularly edged, rounded, flat colonies.

[0036] The 16S rDNA sequence of strain HY-B was determined using primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′, SEQ ID NO: 1) and 1492R (5′-GGTTACCTTGTTACGACTT-3′, SEQ ID NO: 2).

[0037] The determined 16S rDNA sequence of strain HY-B is shown in SEQ ID NO: 3.

[0038] 16S rDNA sequence comparison confirmed that strain HY-B is a Pseudomonas pseudoalcaligenes strain and was designated Pseudomonas pseudoalcaligenes. Strain HY-B was deposited with the General Microbiology Center of the China General Culture Collection Administration (CGMCC) in Beijing, China, on May 19, 2025, under the accession number CGMCC No. 34593.

[0039] 3. Salt tolerance test of Pseudomonas pseudoalcaligenes HY-B

[0040] A single colony of Pseudomonas pseudoalcaligenes HY-B was picked and inoculated into LB liquid medium. The culture was incubated in a constant temperature shaker at 30°C and 160 rpm for 10 h. The bacterial solution was then diluted to 10% with a 0.9 wt% sterile sodium chloride solution. 7 CFU / mL, and then spot-smeared in a triangular shape on LB solid culture medium containing 0g / L, 10g / L, 30g / L, 50g / L, 70g / L, 90g / L, 100g / L, 120g / L, and 150g / L NaCl, 10μL per spot, and 3 spots in each salinity gradient. The cells were cultured in a constant temperature incubator at 30℃ for 24h, and the growth of the colonies was observed to determine the salt tolerance of Pseudomonas pseudoalcaligenes HY-B.

[0041] The growth of Pseudomonas pseudoalcaligenes HY-B on LB solid medium is shown in Table 1.

[0042] Table 1 Growth of Pseudomonas pseudoalcaligenes HY-B on LB solid medium

[0043]

[0044] As shown in Table 1, the salt tolerance range of Pseudomonas pseudoalcaligenes HY-B is 0-90 g / L NaCl, which means that it has a strong salt tolerance.

[0045] 4. Detection of plant growth promotion indicators of Pseudomonas pseudoalcaligenes HY-B

[0046] 1. The ability of Pseudomonas pseudoalcaligenes HY-B to produce the plant hormone indoleacetic acid

[0047] Indole-3-acetic acid (IAA) is an important signaling substance that regulates plant growth and development. It can promote the growth of plant roots, stems, and leaves and accelerate cell division. It is an important type of plant hormone.

[0048] Pseudomonas alcaligenes HY-B was inoculated into LB liquid medium containing L-tryptophan (final concentration 200 mg / L) to an initial concentration of 10 8CFU / mL was measured by incubating the cells in a thermoshaker at 30°C and 160 rpm. Daily, 1 mL of the bacterial culture was centrifuged at 8000 rpm and 4°C for 1 minute. 500 µL of the supernatant was collected and added to 50 µL of Salkowski colorimetric solution (the volume ratio of Salkowski colorimetric solution A to solution B should be 50:1. Mix and prepare immediately before use). The cells were incubated in the dark for 30 minutes, and the absorbance at 530 nm was measured. Simultaneously, 500 µL of LB liquid medium was added to 50 µL of Salkowski colorimetric solution as a control. A standard curve was prepared using the same method using the standard IAA. The amount of IAA produced by Pseudomonas pseudoalcaligenes HY-B was calculated based on the IAA standard curve.

[0049] The calculation results of IAA production by Pseudomonas pseudoalcaligenes HY-B are shown in Figure 2 .

[0050] Depend on Figure 2 It can be seen that Pseudomonas pseudoalcaligenes HY-B has a strong ability to produce the plant hormone IAA.

[0051] 2. Ability of Pseudomonas pseudoalcaligenes HY-B to dissolve inorganic phosphorus

[0052] Phosphorus in soil is divided into available and unavailable forms, depending on whether it can be absorbed and utilized by plants. In salinized soils, phosphate ions react with calcium ions to form inorganic phosphorus precipitates, becoming fixed and unavailable. Certain bacteria can dissolve inorganic phosphorus, converting unavailable phosphorus in the soil into available phosphorus that can be absorbed by plants, thereby promoting plant growth.

[0053] Take 10 μL of Pseudomonas pseudoalcaligenes HY-B bacterial solution and apply it in a triangular shape on a salt (NaCl)-free inorganic phosphorus solid culture medium. Culture it in a constant temperature incubator at 30°C for 3 days and observe the phosphorus solubility.

[0054] The solubility of inorganic phosphorus by Pseudomonas pseudoalcaligenes HY-B is shown in Figure 3 .

[0055] like Figure 3 As shown in the figure, an obvious phosphate solubilization zone was observed on the inorganic phosphate solid culture medium without salt (NaCl), indicating that Pseudomonas pseudoalcaligenes HY-B has the ability to solubilize inorganic phosphate.

[0056] 3. Ability of Pseudomonas pseudoalcaligenes HY-B to produce siderophores

[0057] Siderophores are low-molecular-weight organic compounds secreted by microorganisms that possess a strong ability to chelate iron ions (Fe⁺). In saline soils, iron exists primarily as insoluble Fe⁺, limiting its uptake by plant roots. The microbially mediated siderophore-iron transport system can enhance Fe⁺ uptake by plants.

[0058] Take 10µL of Pseudomonas pseudoalcaligenes HY-B bacterial solution and apply it in a triangular shape on the CAS test plate. Incubate in a constant temperature incubator at 30℃ for 48 hours and observe the production of siderophores (bacteria that can secrete siderophores will have a clear orange halo around the colony).

[0059] The siderophore production of Pseudomonas pseudoalcaligenes HY-B is shown in Figure 4 .

[0060] like Figure 4 As shown, Pseudomonas pseudoalcaligenes HY-B formed an obvious orange halo on the CAS detection plate, proving that Pseudomonas pseudoalcaligenes HY-B has the ability to produce siderophores.

[0061] 5. Application of Pseudomonas pseudoalcaligenes HY-B in improving salt stress resistance in corn

[0062] Pseudomonas pseudoalcaligenes HY-B cells were collected, resuspended in sterile saline (0.9% NaCl), centrifuged, washed three times, and then the absorbance value of Pseudomonas pseudoalcaligenes HY-B at 600 nm (OD 600 ) is 0.2 to obtain a bacterial suspension for later use.

[0063] Corn seeds were soaked and disinfected in a 5wt% sodium hypochlorite solution for 8 minutes, then washed three times with sterile distilled water, and then placed in a 30°C constant temperature incubator for 2 days until the seeds sprouted. Seeds with similar germination degrees were selected for potted experiments.

[0064] Grind the field soil through a 10-mesh sieve, mix it evenly with vermiculite in a volume ratio of 1:1, sterilize it, and pack it into culture boxes, 200 g per box.

[0065] The non-salinization group, mild salinization group, moderate salinization group and severe salinization group were set up, and the electrical conductivities were 0.5ms / cm, 1.5ms / cm, 3.5ms / cm and 5.5ms / cm respectively.

[0066] In addition, different salinization groups with bacteria and groups without bacteria were set up. 20 mL of bacterial suspension was added to the groups with bacteria, and 20 mL of sterile saline was added to the groups without bacteria.

[0067] Nine corn seeds were added to each group and planted in a culture box. The cells were cultured under a 16h / 8h light / dark cycle. After 14 days of culture, the growth of the corn seedlings was observed and their wet weight, plant height, and root length were measured.

[0068] The growth of corn seedlings in each group is shown in Figure 5 The measurement results of corn seedling wet weight are shown in Figure 6 The plant height measurement results are shown in Figure 7, the root length measurement results are shown in Figure 8 .

[0069] Depend on Figure 5 、 Figure 6 、 Figure 7 and Figure 8 It can be seen that Pseudomonas pseudoalcaligenes HY-B has a significant promoting effect on the growth of corn seedlings in different salinized soils, among which:

[0070] In non-salinized soil, the wet weight, plant height and root length of maize seedlings added with Pseudomonas pseudoalcaligenes HY-B increased by 43.5%, 16.6% and 72.8% respectively compared with the control group without Pseudomonas pseudoalcaligenes HY-B addition;

[0071] In slightly saline soil, the wet weight, plant height, and root length of maize seedlings treated with Pseudomonas pseudoalcaligenes HY-B increased by 47.5%, 35.2%, and 34.0%, respectively, compared to the control group without Pseudomonas pseudoalcaligenes HY-B.

[0072] In moderately saline soil, the wet weight, plant height, and root length of maize seedlings treated with Pseudomonas pseudoalcaligenes HY-B increased by 65.7%, 24.7%, and 29.5%, respectively, compared to the control group without Pseudomonas pseudoalcaligenes HY-B.

[0073] Especially in severely saline soil, the wet weight, plant height and root length of corn seedlings added with Pseudomonas pseudoalcaligenes HY-B increased by 124.9%, 80.3% and 99.0% respectively compared with the control group without Pseudomonas pseudoalcaligenes HY-B.

[0074] The above results indicate that the Pseudomonas alcaligenes-like bacteria HY-B screened out in the present invention can enhance the salt stress resistance of corn and promote the growth of corn, and is a corn growth-promoting bacterium.

[0075] It should be noted that the above embodiments are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications may be made based on the above description. It is not possible to enumerate all embodiments here. Any obvious variations or modifications arising from the technical solution of the present invention remain within the scope of protection of the present invention.

Claims

1. A strain of Pseudomonas pseudoalcaligenes HY-B, characterized in that: The pseudoalcaligenes HY-B strain was isolated from saline-alkali soil and has strong salt tolerance, with a salinity tolerance range of 0-90 g / L. It can also enhance plant resistance to salt stress and promote plant growth. It was deposited in the General Microbiology Center of the China Culture Collection Administration, Beijing, China, on May 19, 2025, with a deposit number of CGMCC No. 34593.

2. Use of the Pseudomonas pseudoalcaligenes HY-B according to claim 1 in improving salt stress resistance of plants, wherein the plant is corn.

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