Salt-tolerant growth-promoting staphylococcus amber SYF-3 and application thereof
By using Staphylococcus succinus SYF-3, the environmental friendliness and sustainability issues of saline soil improvement were addressed, significantly promoting the growth performance of Arabidopsis thaliana, maize, and sunflower, and enhancing the salt tolerance and growth effect of crops.
Patent Information
- Application Number
- CN202511748848.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-26
- Publication Date
- 2026-02-27
AI Technical Summary
Saline soils in arid inland climate zones severely restrict agricultural development. Existing improvement methods are either environmentally unfriendly or have short-lasting effects, and there is a lack of effective microbial strains adapted to this environment.
We provide a salt-tolerant and growth-promoting Staphylococcus succinus SYF-3 strain, which has the ability to fix nitrogen, solubilize phosphorus and potassium, produce IAA and ammonia, and can be applied to various crops to improve their growth performance under salt stress.
It significantly promoted the growth of Arabidopsis thaliana, maize and sunflower under different salt stresses, and improved the salt tolerance and growth performance of crops, including leaf growth, plant thickening, fresh weight increase and chlorophyll content enhancement.
Smart Images
Figure CN121574864A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbiology, and more particularly to a salt-tolerant, growth-promoting Staphylococcus aureus strain and its applications. Background Technology
[0002] In inland arid climate zones, scarce rainfall and high evaporation rates lead to the easy accumulation of salts in the soil. Simultaneously, the terrain, characterized by high mountains surrounding a central basin, allows groundwater to easily rise to the surface via capillary action, leaving salts trapped in the topsoil after evaporation. In other areas, groundwater often contains abundant soluble salts; as this groundwater flows to the surface, salts are deposited along with it. Agricultural production is highly dependent on irrigation, and improper irrigation methods (such as over-irrigation and flooding) can cause groundwater levels to rise, leading to the accumulation of soluble salts in the surface layer and secondary salinization. These factors severely restrict agricultural development.
[0003] Currently, methods for improving saline-alkali soils mainly include physical methods such as underground drainage and flushing, chemical methods such as gypsum and humic acid, and biological methods such as microbial agents and planting salt-tolerant plants. Biological improvement is an environmentally friendly and long-lasting method that improves saline-alkali soils through the growth and metabolism of organisms. Finding effective microbial strains adapted to the ecological environment of arid inland climate zones has become a crucial part of agricultural production. Summary of the Invention
[0004] The purpose of this invention is to provide a salt-tolerant, growth-promoting bacterium, *Staphylococcus aureus*, which possesses nitrogen fixation, phosphorus solubilization, potassium solubilization, and IAA and NH4 production capabilities. + It has various effects and can be used to promote the growth of a variety of crops.
[0005] The Staphylococcus succinus SYF-3 of this invention is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, on December 3, 2024, with accession number CCTCC NO: M 20242699.
[0006] This invention provides the application of Staphylococcus aureus SYF-3 in nitrogen fixation.
[0007] This invention provides the application of Staphylococcus aureus SYF-3 in phosphorus solubilization.
[0008] This invention provides the application of Staphylococcus aureus SYF-3 in potassium solubilization.
[0009] This invention provides the application of Staphylococcus aureus SYF-3 in the secretion of plant growth hormone IAA.
[0010] This invention provides the application of Staphylococcus aureus SYF-3 in ammonia production.
[0011] This invention provides the application of Staphylococcus aureus SYF-3 in salt tolerance.
[0012] This invention provides the application of Staphylococcus aureus SYF-3 in promoting plant growth under salt stress.
[0013] Furthermore, the plants mentioned are Arabidopsis thaliana, maize, and sunflower.
[0014] The beneficial effects of this invention are:
[0015] The *Staphylococcus aureus* SYF-3 of this invention possesses nitrogen-fixing, phosphorus-solubilizing, and potassium-solubilizing abilities, which can promote crop nitrogen absorption and increase the content of available phosphorus and potassium in the soil. It also has the ability to secrete plant growth hormone IAA and produce ammonia.
[0016] The *Staphylococcus aureus* SYF-3 strain of this invention exhibits significant growth-promoting effects on Arabidopsis thaliana, maize, and sunflower under different salt stress conditions. Specifically, when the crop is Arabidopsis thaliana, *Staphylococcus aureus* SYF-3 enhances its salt tolerance and promotes leaf growth under salt-free, 50 mM NaCl, and 100 mM NaCl stress conditions. When the crop is maize, under salt-free and 200 mM NaCl stress conditions, *Staphylococcus aureus* SYF-3 promotes plant growth, increases stem thickness, overall plant fresh weight, and leaf area, and significantly increases the content of proline, malondialdehyde (MDA), and soluble sugars in the plant. When the crop is sunflower, under salt-free and 200 mM NaCl stress conditions, *Staphylococcus aureus* SYF-3 increases plant height, fresh weight, stem diameter, leaf area, number of leaves, and chlorophyll content, and also increases the content of MDA and SOD in the sunflower. Attached Figure Description
[0017] Figure 1 The colony morphology of Staphylococcus succinus SYF-3;
[0018] Figure 2 The cell morphology of Staphylococcus succinus SYF-3;
[0019] Figure 3 Phylogenetic tree of Staphylococcus succinus SYF-3;
[0020] Figure 4 Results of the activity-promoting assay for Staphylococcus succinus SYF-3;
[0021] Figure 5The effect of Staphylococcus succinus SYF-3 on Arabidopsis thaliana growth;
[0022] Figure 6 The effect of Staphylococcus succinus SYF-3 bacterial inoculation on maize (potted) growth 60 days after inoculation;
[0023] Figure 7 The effect of Staphylococcus succinus SYF-3 bacterial inoculation on the growth of sunflowers (potted) 60 days after inoculation. Detailed Implementation
[0024] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.
[0025] Example 1:
[0026] The Staphylococcus succinus in this embodiment is Staphylococcus succinus SYF-3, which has been deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, on December 3, 2024, with accession number CCTCC NO: M 20242699.
[0027] The isolation method for Staphylococcus succinus SYF-3 in this embodiment is as follows:
[0028] Take 5.0 g of soil sample from Xinjiang mushroom cultivation areas and place it in 100 mL of sterile water. Incubate at 28°C and 120 rpm with shaking for 30 min. Take 1 mL of the supernatant and perform serial dilutions to a final concentration of 10. -5 Dilute to 10. -2 10 -3 10 -4 10 -5 100 μL of each of the diluted bacterial suspensions were spread onto LB agar medium containing 80 g / L NaCl using the dilution plating method. The culture was inverted at 28 °C for 24 h. Single colonies obtained from isolation and purification were picked and inoculated onto LB agar slant medium and stored at 4 °C for later use. This is strain SYF-3.
[0029] Example 2: Identification of strain SYF-3
[0030] The colony morphology of strain SYF-3 is as follows: Figure 1 As shown, the bacterial cell morphology is as follows Figure 2As shown. The colonies are round, white, with a raised center, a dry surface, and a rough periphery; strain SYF-3 is a Gram-positive, short rod-shaped, non-motile bacterium.
[0031] Physiological and biochemical assays were performed on strain SYF-3. The sugar fermentation test showed it could ferment glucose and maltose, but not sucrose or lactose; the indole test, starch hydrolysis test, methyl red test (MR test), VP test, citrate utilization test, hydrogen sulfide production test, and urease test were all negative. It could still grow at a salinity of 20% and at temperatures between 20 and 40°C. According to Bergey's Manual of Bacteriological Identification, this strain possesses the biochemical characteristics of Staphylococcus.
[0032] Genomic DNA was extracted from strain SYF-3 using the CTAB method. The 16S rDNA of the strain was amplified by PCR using primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR amplification system consisted of: 1 μL DNA template, 10 μL 2×Taq PCR Master Mix, 1 μL 5 μmol / L 27F, 1 μL 5 μmol / L 1492R, and 12 μL ddH2O, for a total of 25 μL. The PCR amplification conditions were: 94℃ for 5 min; 94℃ for 45 s, 55℃ for 45 s, 72℃ for 1 min, for a total of 30 cycles; 72℃ for 10 min. The 16S rDNA sequence (1473 bp) of strain SYF-3 is shown in SEQ ID NO.1 of the sequence listing. The 16S rDNA sequence of strain SYF-3 was aligned to the GenBank database, and a phylogenetic tree of strain SYF-3 was constructed using Mega7 software. The results are as follows: Figure 3 As shown in the figure. The results showed that strain SYF-3 had a sequence similarity of 98.74% with the type strain Staphylococcus succinus 16S (KU550582). Based on the combined morphological observation and physiological and biochemical results, strain SYF-3 was identified as Staphylococcus succinus. The sequence of strain SYF-3 has been uploaded to NCBI, and the accession number is MK583490.
[0033] Example 3: Detection of the activity-promoting properties of Staphylococcus succinus SYF-3
[0034] The activated Staphylococcus succinus SYF-3 was inoculated into Assumption nitrogen-free medium, Monkina inorganic phosphorus medium, Monkina organic phosphorus medium, potassium-solubilizing screening medium and 8% NaCl beef extract peptone medium to observe the growth of the strain.
[0035] The IAA production activity of the strain was detected by inoculating *Staphylococcus succinus* SYF-3 into LB liquid medium supplemented with 100 mg / L tryptophan and incubating at 28°C for 48 h. The medium was then mixed with Salkowski colorimetric solution for reaction, and the mixture was incubated in the dark for 30 min. The color of the liquid was observed to determine whether the strain possessed the ability to produce IAA. The NH4 production of the strain was also assessed. + To detect activity, Staphylococcus succinus SYF-3 was inoculated into peptone aqueous medium and cultured at 28°C for 5 days. A 1:1 volume ratio of bacterial culture to Nessler's reagent was used to observe whether a red precipitate formed, thus preliminarily determining whether the strain possessed NH4+ production capabilities. + The activity of Staphylococcus succinus SYF-3 was detected. The results of the activity assay are shown in Table 1. Figure 4 As shown.
[0036] The results showed that *Staphylococcus succinus* SYF-3 grew well on Assumption nitrogen-free medium, potassium-solubilizing selection medium, and 8% NaCl beef extract peptone medium. *Staphylococcus succinus* SYF-3 also grew well on Monkina inorganic phosphorus medium and Monkina organic phosphorus medium, exhibiting a clear zone. The culture medium of *Staphylococcus succinus* SYF-3 turned pink to orange-red after being mixed with Salkowski's colorimetric solution and Nessler's reagent. The results demonstrated that *Staphylococcus succinus* SYF-3 possesses nitrogen-fixing, phosphorus-solubilizing, potassium-solubilizing, and salt-tolerant (8%) activities, and is capable of secreting IAA and producing ammonia.
[0037] Table 1. Results of Staphylococcus succinus SYF-3 activating assay
[0038]
[0039] The Assumption nitrogen-free medium formula is as follows: mannitol 10g, MgSO4•7H2O 0.2g, CaSO4•2H2O 0.1g, KH2PO4 0.2g, CaCO3 5g, agar 20g, pH 7.2~7.4, and distilled water is added to a final volume of 1L.
[0040] The formula for Monkina Inorganic Phosphorus Medium is as follows: 10g glucose, 0.5g (NH4)2SO4, 0.3g KCl, 0.3g NaCl, 0.03g FeSO4·7H2O, 0.3g MgSO4·7H2O, 0.03g MnSO4·4H2O, 5g Ca3(PO4)2, 18g agar powder, pH 7.0~7.5, and distilled water to a final volume of 1L.
[0041] The formula for Mongkina's organic phosphorus culture medium is as follows: 10g glucose, 0.5g (NH4)2SO4, 0.3g KCl, 0.3g NaCl, 0.03g FeSO4·7H2O, 0.3g MgSO4·7H2O, 0.03g MnSO4·4H2O, 5g CaCO3, 0.2g lecithin, 18g agar powder, pH 7.0~7.5, and distilled water to a final volume of 1L.
[0042] Potassium-solubilizing screening medium formula: Na2HPO4 2g, FeCl3 0.005g, CaCO3 0.1g, MgSO4•7H2O 0.5g, potassium feldspar powder 1g, sucrose 5g, agar 18g, pH 7.5, add distilled water to a final volume of 1L.
[0043] Peptone aqueous culture medium formula: 20g peptone, 50g NaCl, add distilled water to a final volume of 1L.
[0044] Formula for 8% NaCl beef extract peptone medium: 10g peptone, 80g NaCl, 3g beef extract, 20g agar, pH 7.2~7.4, add distilled water to a final volume of 1L.
[0045] Example 4: Verification of the growth-promoting effect of Staphylococcus succinus SYF-3
[0046] (1) Growth-promoting effect of Staphylococcus succinus SYF-3 on Arabidopsis thaliana
[0047] Arabidopsis seeds were washed three times with sterile water to remove floating, inferior seeds. They were then soaked in 70% ethanol for 30 seconds, immediately followed by four washes with sterile water to remove residual ethanol. Finally, they were soaked in 0.2% sodium hypochlorite for 5 minutes, followed by four washes with sterile water. After surface sterilization, 16 seeds were planted approximately 2.5 cm from the center of each plate using a 200 μL pipette. The plates were incubated for 5 days in a 28°C incubator with 16 hours of darkness and 8 hours of light. Once the Arabidopsis had germinated and developed two leaves, strain Staphylococcus succinus SYF-3 was selected and inoculated onto the central LB medium. The same procedure was performed, with sterile water used as the control (CK). Each treatment included salinity gradients of 0, 50, and 100 mM, and was performed in triplicate. Growth was observed after 30 days.
[0048] The results are as follows Figure 5 As shown: Staphylococcus succinus SYF-3 significantly promoted Arabidopsis leaf growth compared to the control under salt-free stress and 50 mM NaCl stress, promoted Arabidopsis seed germination under 100 mM NaCl, and improved Arabidopsis salt tolerance, but ultimately died due to the high salt concentration.
[0049] (2) Growth-promoting effect of Staphylococcus succinus SYF-3 on maize
[0050] Preparation of bacterial culture: Staphylococcus succinus SYF-3 was inoculated into 100 mL of liquid LB medium and incubated on a shaker at 28 °C until the bacterial concentration reached 10⁻⁶. 8 CFU / mL.
[0051] Seed treatment: Seeds purchased from the farmers' market (corn variety Nongke Yu 368) were soaked in saturated saline solution to remove floating and damaged seeds. Only plump, intact seeds of similar size were selected. The seeds were rinsed three times with sterile water, disinfected with 70% ethanol for 1 minute, immediately rinsed five times with sterile water, then soaked in 0.5% sodium hypochlorite solution for 10 minutes, rinsed five times with sterile water, and placed on moist filter paper in the dark overnight until the seeds showed signs of sprouting. The sprouted seeds were then soaked in 10... 8 The seeds were soaked in bacterial solution at CFU / mL for 4 hours and then left to use; the seeds soaked in sterile water served as the control group (CK).
[0052] Pot experiment: Uniformly growing maize seedlings were transplanted into pots containing a nutrient soil:vermiculite:perlite (v / v) ratio of 3:1:1. Sterile water was applied every 5 days, ensuring the soil was thoroughly soaked but not overflowing. Four seedlings were planted per pot, with six replicates per treatment. Subsequent treatments were administered after 7 days of acclimatization. Salt treatment: Maize was treated with a 0 and 200 mM NaCl salt gradient, with root irrigation applied along with each bacterial treatment. Bacterial solution treatment: 300 mL / pot was applied every 5 days, with sterile water used as the control group (CK). Ten replicates were applied to each treatment. Data were collected after 60 days of growth at room temperature. The effects of Staphylococcus succinus SYF-3 on maize (potted) growth are as follows: Figure 6 As shown, Staphylococcus succinus SYF-3 significantly promoted the growth of maize (potted plants) in both salt-free and 200mM NaCl salt stress conditions, increasing plant height, stem diameter, leaf area, and number of leaves.
[0053] The plants were harvested, and the root length, plant height, fresh weight, chlorophyll content, proline, soluble sugar, and SOD content of maize were measured as evaluation indicators of the growth-promoting effect of strain Staphylococcus succinus SYF-3. The results are shown in Table 2.
[0054] Table 2. Effects of different NaCl concentrations on maize growth of Staphylococcus succinus SYF-3.
[0055]
[0056] Note: CK represents the control group, and N represents the percentage increase in the treatment group compared to CK.
[0057] From Table 2 and Figure 6 It was found that, compared with the control group, Staphylococcus succinus SYF-3 increased maize plant height by 17.04%, stem diameter by 45.05%, and underground and above-ground fresh weight by 39.92% and 42.05%, respectively, and chlorophyll content by 13.17% when treated with 0 mM NaCl. After treatment with 200 mM NaCl, the maize plant height of strain SYF-3 increased by 32.35%, stem diameter by 11.03%, and underground and above-ground fresh weight by 6.42% and 59.98%, respectively, malondialdehyde content increased by 95.35%, proline content increased by 190.04%, and soluble sugar and SOD increased by 83.76% and 97.19%, respectively.
[0058] (3) The growth-promoting effect of Staphylococcus succinus SYF-3 on sunflowers
[0059] Preparation of bacterial culture: Staphylococcus succinus SYF-3 was inoculated into 100 mL of liquid LB medium and incubated on a shaker at 28 °C until the bacterial concentration reached 10⁻⁶. 8 CFU / mL.
[0060] Seed treatment: Sunflower seeds (variety: Dwarf Sunflower) purchased from the farmers' market were soaked in saturated salt water to remove floating and damaged seeds. Only plump, intact seeds of similar size were selected. The seeds were then rinsed three times with sterile water, disinfected with 70% ethanol for 1 minute, immediately rinsed five times with sterile water, and then soaked in 0.5% sodium hypochlorite solution for 10 minutes. After rinsing five times with sterile water, the seeds were placed on moist filter paper and left in the dark overnight until they began to sprout. The sprouted seeds were then soaked in 10... 8 Seeds soaked in CFU solution for 4 hours were ready for use; seeds soaked in sterile water served as the control group (CK).
[0061] Pot Experiment: Uniformly growing sunflower seedlings were transplanted into pots containing a nutrient soil:vermiculite:perlite (v / v) ratio of 3:1:1. Sterile water was applied every 5 days, ensuring the soil was thoroughly moistened without overflowing. Four seedlings were planted per pot, with six replicates per treatment. Subsequent treatments were administered after 7 days of acclimatization. Salt Treatment: Sunflowers were treated with a 0 and 200 mM NaCl salt gradient, with root irrigation applied along with each inoculum treatment. Inoculum Treatment: Treatment was administered every 5 days, 300 mL / pot. Sterile water served as the control group (CK). Ten replicates were applied to each treatment. Data were collected after 60 days of growth at room temperature. The effects of Staphylococcus succinus SYF-3 on the growth of potted sunflowers are as follows: Figure 7 As shown, Staphylococcus succinus SYF-3 significantly promotes the growth of sunflower (potted) in both salt-free and 200mM NaCl salt stress conditions, increasing plant height, stem diameter, leaf area, and number of leaves.
[0062] Plants were harvested, and the root length, plant height, fresh weight, chlorophyll content, proline, soluble sugar, and SOD content of sunflowers were measured as evaluation indicators of the growth-promoting effect of strain Staphylococcus succinus SYF-3. The results are shown in Table 3.
[0063] Table 3. Effects of different NaCl concentrations on sunflower growth of Staphylococcus succinus SYF-3
[0064]
[0065] Note: CK represents the control group, and N represents the percentage increase in the treatment group compared to CK.
[0066] From Table 3 and Figure 7 It was found that, compared with the control group, the height of sunflower plants treated with Staphylococcus succinus SYF-3 increased by 20.26%, stem diameter increased by 4.95%, and the underground and above-ground fresh weights increased by 9.68% and 33.26%, respectively, while the chlorophyll content increased by 0.18%. After treatment with 200mM NaCl, the height of sunflower plants treated with strain SYF-3 increased by 31.01%, stem diameter increased by 17.24%, and the underground and above-ground fresh weights increased by 43.41% and 61.12%, respectively, while the proline content increased by 190.04%, and the soluble sugar and SOD increased by 51.08% and 97.15%, respectively.
[0067] The *Staphylococcus aureus* SYF-3 strain of the present invention possesses nitrogen-fixing, phosphorus-solubilizing, and potassium-solubilizing abilities, and produces IAA and NH4. + The Staphylococcus succinus SYF-3 described in this invention has growth-promoting effects on various plants. It can promote the leaf growth of Arabidopsis thaliana under salt-free stress and 50mM NaCl stress, improve the salt tolerance of Arabidopsis thaliana and promote its germination under 100mM NaCl stress, and promote the root growth of maize and sunflower under salt-free stress and 200mM NaCl, increasing fresh weight and promoting proline accumulation.
Claims
1. A salt-tolerant growth-promoting Staphylococcus succinii SYF-3, characterized in that, The Staphylococcus succinus SYF-3 is Staphylococcus succinus SYF-3, which has been preserved in the China Center for Type Culture Collection, Wuhan University, Wuhan City, on December 3, 2024, with the preservation number CCTCC NO: M 20242699.
2. The Staphylococcus succinus SYF-3 of claim 1 in nitrogen fixation.
3. The Staphylococcus succinus SYF-3 of claim 1 in phosphorus solubilization.
4. The Staphylococcus succinus SYF-3 of claim 1 in potassium solubilization.
5. The Staphylococcus succinus SYF-3 of claim 1 in secreting plant growth hormone IAA.
6. The Staphylococcus succinus SYF-3 of claim 1 in ammonia production.
7. The Staphylococcus succinus SYF-3 of claim 1 in salt tolerance.
8. The Staphylococcus succinus SYF-3 of claim 1 in promoting plant growth under salt stress.
9. Use according to claim 8, characterized in that, The plant is Arabidopsis thaliana, corn and oil sunflower.