Sphingomonas sp. And fungicide thereof and application of spingomonas sp. In saline-alkali soil treatment

Abstract

The invention discloses a sphingomonas sphingomonas, a bacterial agent thereof and application of the sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas sphingomonas The sphingomonas disclosed by the invention has excellent saline-alkaline tolerance, has the functions of fixing nitrogen, decomposing organic phosphorus and producing ACC deaminase, breaks through the limitation of single function of a traditional strain, and has a very good application prospect in saline-alkaline soil improvement, plant growth promotion and ecological restoration.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Sphingomonas strain and a bacterial agent thereof and application in saline-alkali land treatment. Background Art

[0002] Globally, saline-alkali land covers over 950 million hectares, and in my country, approximately 100 million hectares, with an expanding trend. This severely constrains agricultural production and the ecological environment. The core problem of saline-alkali land is the deterioration of soil physical and chemical properties (such as compaction and poor air and water permeability) and reduced biological activity (such as imbalanced microbial community structure and obstructed recycling of key nutrients) caused by high salinity and high pH (alkaline stress). This leads to suppressed plant growth, low productivity, and ecological degradation. While traditional physical and chemical amelioration methods (such as irrigation to remove salt, application of gypsum, and chemical amendments) have some effectiveness, they generally suffer from high costs, potential for secondary pollution, and short-term effects.

[0003] In recent years, the use of salt- and alkali-tolerant microorganisms for bioremediation has been regarded as an important direction for saline-alkali land management due to its advantages such as environmental friendliness and strong sustainability. However, the microbial strains used in the existing technology often have problems such as single function, limited salt- and alkali tolerance, or a significant decrease in activity under salt- and alkali stress. This leads to the need to mix and apply multiple functional strains in practical applications, which not only increases the application cost and complexity, but also may cause antagonism between different strains, affecting the overall effect. Therefore, there is an urgent need to discover or construct new multifunctional microbial strains that have both excellent salt- and alkali-tolerant properties and multiple plant growth-promoting functions, and to develop efficient and stable microbial agents to overcome the limitations of existing technologies. Summary of the Invention

[0004] To address the deficiencies of the prior art, the present invention aims to provide a Sphingomonas strain and its bacterial agent and its application in saline-alkali land treatment. The Sphingomonas strain of the present invention has excellent salt-alkali tolerance and has the functions of nitrogen fixation, organic phosphorus decomposition and ACC deaminase production.

[0005] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0006] A strain of Sphingomonas sp., named R-3, was deposited in the General Microbiology Center of the China Culture Collection Administration on May 30, 2025, and the deposit number of the strain is: CGMCC No. 34720.

[0007] A bacterial agent carrier produced by Sphingomonas.

[0008] The invention discloses an application of a bacterial agent or a bacterial agent carrier in nitrogen fixation in saline-alkali land.

[0009] Application of a bacterial agent or a bacterial agent carrier in degrading organic phosphorus in saline-alkali soil.

[0010] Application of a bacterial agent or a bacterial agent carrier in producing ACC deaminase.

[0011] A preparation method of a bacterial agent carrier, comprising the following specific steps:

[0012] S1, fermentation liquid treatment: inoculating sphingomonas R-3 into a culture medium, centrifuging, and obtaining a fermentation liquid;

[0013] S2, embedding granulation: mixing the fermentation liquid with a sodium alginate solution, adding bentonite, cross-linking and solidifying, and hot air drying to obtain the bacterial agent carrier.

[0014] Preferably, in the foregoing step S1, the formula of the culture medium is: 20 g / L of glucose, 10 g / L of tryptone, 1% of NaCl, 0.3% of L-tryptophan, and pH 7.0; the culture conditions are: 25-35 DEG C, 160-200 rpm, and fermentation for 55-65 h; and the centrifugation conditions are: 7000-9000 rpm, and 8-12 min.

[0015] Preferably, in the foregoing step S2, the volume ratio of the fermentation liquid to the sodium alginate solution is 1:2-4.

[0016] The sphingomonas of the present application has excellent salt-alkali tolerance, and has the functions of nitrogen fixation, organic phosphorus degradation, and ACC deaminase production, thereby breaking through the limitation of single function of traditional strains, and having a good application prospect in saline-alkali soil improvement, plant growth promotion, and ecological restoration. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is a phylogenetic tree of the sphingomonas in the present application;

[0018] Figure 2 is a colony morphology diagram of the sphingomonas in the present application. DETAILED DESCRIPTION

[0019] The present application will be specifically introduced below in combination with the drawings and specific examples.

[0020] Example 1, isolation and identification of the strain:

[0021] Plant rhizosphere soil was collected from a saline-alkali soil in Dongtai Town, Yancheng City, Jiangsu Province, and after removing surface soil, large soil blocks, and rotten leaves, the soil was loaded into a sterile bag and transported back to the laboratory, 0.1 g of the test soil sample was added into 900 muL of sterilized deionized water, and after mixing and oscillation, a soil suspension was obtained by standing for 2-3 min, 100 muL of the soil suspension was taken and added into 900 muL of sterilized deionized water to obtain a 10 -1g / mL soil suspension, and so on for gradient dilution to obtain 10 -2 g / mL, 10 -3 g / mL, 10 -4 g / mL and 10 -5 g / mL soil suspension, vortex to mix thoroughly, and then let it stand. Take 100 μL of each concentration gradient soil suspension and spread it onto LB solid medium (pH 9, 5% NaCl) using a spreading rod. Label the culture plates with the date and invert them in a 30°C incubator for 7 days. After 7 days, select individual colonies with distinct morphology, color, and size and inoculate them into liquid medium for culture. Then, streak the culture medium. Repeat this streak operation at least three times until a single colony is obtained. Record the colony morphology. Mix the purified strains with glycerol and store at -80°C until needed.

[0022] The present invention determined the 16S rDNA sequence of the strain. Universal bacterial primers were used for bacterial amplification. The forward primer was Eubac27F: (5-AGAGTTTGATC-CTGGCTCAG-3), and the reverse primer was Eubac1492R: (5-GGTTACCTTGTTACGACTT-3). 25 μL of 2×Mix Taq was mixed with 2 μL each of the forward and reverse primers and 2 μL of bacterial solution, and then water was added to a 50 μL system for PCR amplification. The PCR reaction procedure was 94°C pre-denaturation for 4 minutes; 94°C denaturation for 30 seconds; 55°C annealing for 1 minute; 72°C extension for 1.5 minutes, for a total of 30 cycles; and 72°C extension for 10 minutes. The PCR product was detected by 1% agarose gel electrophoresis and then sent to a sequencing company for sequencing. The 16S rDNA sequence of the strain was searched and compared against the NCBI database using the BLAST method. The 16S rDNA sequences of strain R-3 and its related strains were aligned using Mega (Version 7.0) software, and the phylogenetic tree was constructed using the Neighbor-joining method, as follows Figure 1 .

[0023] The bacteria were identified as Sphingomonas and named R-3. Morphological characteristics: Gram-negative short rods with orange-yellow colonies and smooth surface (such as Figure 2 shown).

[0024] The above-mentioned strain was deposited in the General Microbiology Center of the China Culture Collection Administration on May 30, 2025. Its classification name is Sphingomonas sp., the strain name is R-3, the storage address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the strain collection number is: CGMCC No. 34720.

[0025] Example 2, a method for preparing a Sphingomonas agent carrier, comprising the following specific steps:

[0026] (1) Fermentation broth treatment: Sphingomonas R-3 was inoculated into an optimized culture medium (glucose 20 g / L, tryptone 10 g / L, NaCl 1%, pH 7.0) containing 0.3% L-tryptophan, and fermented at 30°C, 180 rpm for 60 hours. The supernatant was collected by centrifugation (8000 rpm, 10 min), and a fermentation broth with an IAA concentration of 50±2.1 μg / mL was obtained.

[0027] (2) Embedding and granulation:

[0028] Material ratio: The fermentation broth was mixed with 4% sodium alginate solution at a ratio of 1:3 (v / v), and 5% bentonite was added (to improve the stability of the particles).

[0029] Cross-linking and solidification: The mixed solution was dropped into a 2% CaCl2 solution using a syringe, and magnetic stirring (200 rpm) was performed for 30 minutes to form gel particles with a particle size of 2-3 mm.

[0030] Drying and storage: Hot air drying was performed at 40°C until the water content was ≤8%, and the agent carrier was obtained. It was sealed and stored in the dark (the survival rate of live bacteria was >90% at 4°C for 6 months).

[0031] Example 3, the effect of NaCl concentration on the growth of strain R-3:

[0032] Strain R-3 was inoculated into LB liquid medium and incubated in a constant temperature incubator at 30°C, 180 rpm for 16 hours for activation. Then, 1 mL of activated seed liquid was inoculated into 100 mL of LB liquid medium containing different concentrations of sodium chloride (0 g / L, 10 g / L, 30 g / L, 50 g / L, 70 g / L, 90 g / L, 100 g / L) with a pH value of 8. The components of the LB liquid medium were: yeast extract 5 g, tryptone 10 g, NaCl 5 g, water 1000 mL. 10 μL of the diluted liquid was spotted on each plate 3 times, and then placed in a constant temperature incubator at 30°C for inverted culture for 24 hours. The growth of the colonies was observed to determine the salt tolerance of strain R-3. The growth of strain R-3 on the above LB solid medium is shown in Table 1.

[0033] Table 1 Growth of strain R-3 on LB solid medium containing different concentrations of sodium chloride

[0034]

[0035]

[0036] From Table 1, the highest salt concentration that strain R-3 can tolerate is 90 g / L, and it has strong salt tolerance.

[0037] Example 4, Effect of Alkali Concentration on Growth of Strain R-3:

[0038] Strain R-3 was inoculated into LB liquid medium and incubated in a 30°C, 180 rpm constant temperature incubator for 16 hours for activation, then the bacterial solution was collected and diluted 10 times with sterile 0.9% sodium chloride solution, 10 μL of the diluted solution was spotted on LB solid medium with different pH values (7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, adjusted with sodium hydroxide solution), 10 μL of the diluted solution was spotted on each plate 3 times, then placed in a 30°C constant temperature incubator and incubated for 24 hours, the growth of the colonies was observed, and the alkali tolerance of strain R-3 was determined. The growth of strain R-3 on the above LB solid medium is shown in Table 2.

[0039] Table 2 Growth of Strain R-3 on LB Solid Medium Containing Different Concentrations of Sodium Hydroxide

[0040] pH value Colony growth 7.0 Colonies grew at 3 points, 3.0-3.6 mm in diameter 7.5 Colonies grew at 3 points, 3.1-3.7 mm in diameter 8.0 Colonies grew at 3 points, 3.0-3.6 mm in diameter 8.5 Colonies grew at 3 points, 3.0-3.7 mm in diameter 9.0 Colonies grew at 3 points, 3.1-3.5 mm in diameter 9.5 Colonies grew at 3 points, 2.1-2.4 mm in diameter 10.0 No colonies grew at 3 points

[0041] From Table 2, the highest pH value that strain R-3 can tolerate is 9.5, and it has strong alkali tolerance.

[0042] Example 5, Verification and Optimization of IAA (Indole Acetic Acid) Production by Strain R-3:

[0043] Activated strain R-3 was inoculated into optimized medium containing 0%, 0.1%, 0.3%, 0.5% L-tryptophan (glucose 20 g / L, tryptone 10 g / L, NaCl 1%, pH 7.0) and cultured at 30°C, 180 rpm for 0-96 hours (sampling every 24 hours). Salkowski colorimetric method was used to determine the concentration of IAA: 1 mL of the fermentation broth was centrifuged at 12000 rpm for 5 minutes, and the supernatant was taken. 2 mL of Salkowski reagent (0.5 M FeCl3+35% HCIO4, 1:50) was added. The reaction was carried out in the dark for 30 minutes, and the absorbance at 530 nm was detected. The specific results are shown in Tables 3 and 4.

[0044] Table 3 Relationship between IAA Yield and Tryptophan Concentration

[0045] L-tryptophan concentration (%) Maximum IAA yield (pg / mL) Time to peak (h) 0 4.8±1.2 96 0.1 28.5±1.2 72 0.3 52.7±2.1 60 0.5 48.9±1.8 48

[0046] Table 4 Fermentation Kinetics Curve (0.3% Tryptophan)

[0047] Culture time (hours) <![CDATA[OD 600 ]]> IAA (pg / mL) Culture medium pH 0 0.1 0 7.0 24 0.9 12.3 6.8 48 1.6 45.2 6.5 72 1.8 52.7 6.3

[0048] As shown in Tables 3 and 4, IAA synthesis was accompanied by a decrease in the pH of the culture medium (7.0→6.3), which indicated that the acidic environment may inhibit the activity of the late bacteria. Therefore, strain R-3 is suitable for use in saline-alkali land.

[0049] Example 6: Verification of the plant growth-promoting ability of strain R-3:

[0050] Treatment group: Added R-3 fermentation broth (IAA 50 μg / mL). Control group: No IAA added. The results of the wheat hydroponic experiment after 7 days are shown in Table 5.

[0051] Table 5 Wheat hydroponic experimental results

[0052] Index Control group Treatment group Root length (cm) 5.2 9.8 Lateral root number (pieces) 4.1 8.7

[0053] The above experiments showed that in the absence of exogenous tryptophan, the IAA production was less than 5 μg / mL, indicating that R-3 can synthesize IAA through a tryptophan-dependent pathway.

[0054] Example 7, Verification of the multifunctional properties of strain R-3:

[0055] (1) Determination of nitrogen fixation function of strain R-3:

[0056] Strain R-3 was inoculated into nitrogen-fixing medium (nitrogen-free) consisting of 10 g / L sucrose, 0.2 g / L KH2PO4, 0.2 g / L MgSO4·7H2O, and 6% NaCl, pH 7.2, and cultured at 30°C and 180 rpm for 72 hours. Nitrogenase activity was measured using the acetylene reduction method. The culture was injected into a sealed bottle, injected with 10% acetylene gas, and reacted at 30°C for 24 hours. Ethylene production was determined by gas chromatography (FID detector, Porapak N column). Activity was calculated as 1 nmol C2H4 / h·mg protein. Azospirillum brasilense was used as a positive control. Detailed results are shown in Table 6.

[0057] Table 6 Results of nitrogen fixation function test of strain R-3

[0058] Strain Nitrogenase activity (nmol C2H4 / h mg protein) R-3 15.8±1.2 Positive control 12.3±0.9

[0059] (2) Determination of phosphate solubilization function of strain R-3:

[0060] Strain R-3 was inoculated into NBRIP medium and incubated at 30°C for 5 days. The phosphate-solubilizing medium (NBRIP) consisted of 10 g / L glucose, 5 g / L Ca(3(PO4)2, 0.5 g / L MgCl2, and 0.1 g / L (NH4)2SO4, pH 7.0. The diameter of the phosphate-solubilizing zone and the soluble phosphorus content were determined using the molybdenum blue colorimetric method. The culture supernatant was centrifuged and ammonium molybdate-sulfuric acid reagent was added. After color development, the absorbance at 700 nm was measured. The results are shown in Table 7.

[0061] Table 7 Results of phosphate solubilization function test of strain R-3

[0062] Index R-3 Blank medium Phosphorus solubilizing circle diameter (mm) 8.5±0.3 0 Soluble phosphorus (mg / L) 32.4±1.5 0.2±0.1

[0063] (3) ACC deaminase activity assay:

[0064] Strain R-3 was cultured in ACC medium for 48 hours, harvested by centrifugation, and lysed for α-ketobutyrate production (colorimetric method, λ = 540 nm). ACC deaminase induction medium consisted of DF salts-based medium with 3 mM ACC (alternative nitrogen source), pH 8.0. The lysate was reacted with ACC substrate (30 min, 30°C) and terminated with 0.56 M HCl. α-ketobutyrate production was detected by colorimetric analysis using 2,4-dinitrophenylhydrazine. The results are shown in Table 8.

[0065] Table 8 ACC deaminase activity assay results

[0066] Treatment Alpha-ketobutyric acid (pmol / mg protein) R-3+ACC 4.2±0.3 R-3-ACC 0.1±0.02

[0067] The above results show that strain R-3 has the functions of nitrogen fixation, organic phosphorus decomposition and ACC deaminase production, breaking through the limitation of single function of traditional strains.

[0068] Example 8: Actual effect of bacterial agent carrier in improving saline-alkali land:

[0069] To verify the practical effect of the microbial agent carrier of the present invention in improving saline-alkali land, a field application test was conducted in coastal saline-alkali land in Dongtai, Jiangsu Province (initial soil pH 9.2, conductivity 8.6 dS / m, total salt content 0.8%). The salt-tolerant wheat variety "Yanmai No. 1" was selected for the test, and three treatment groups were set up: T1: blank control (no microbial agent carrier); T2: in-furrow application of the microbial agent carrier (a 10 cm deep furrow was dug before sowing, and the carrier was evenly applied at 30 kg / hectare, followed by covering with soil, with a carrier viable count of ≥1.5×10 9CFU / g); T3: hole-applied inoculant carrier (2 g of carrier / hole mixed with seeds and then sown at sowing time). Rhizosphere soil samples (0-20 cm depth) were collected 90 days after sowing and analyzed for physical, chemical, and microbial indices to evaluate the improvement effect of the carrier on saline-alkali soil and the growth-promoting effect of the inoculant on saline-alkali crops. Specific results are shown in Tables 9 and 10.

[0070] Table 9 Improvement effect of bacterial agent carrier on saline-alkali soil

[0071]

[0072] Note: Data are mean ± standard deviation (n=3), “*” indicates significant difference compared with the control (p<0.05).

[0073] Table 9 shows that the multifunctional carrier of the microbial agent achieved targeted improvement of saline-alkali soils through the synergistic effect of strain R-3. Furrow application (T2) significantly increased rhizospheric ACC deaminase activity, effectively neutralizing alkaline substances and reducing the pH by 0.6 units. Simultaneously, the nitrogen fixation function drove a 52% increase in soil alkaline nitrogen, and the phosphorus solubility function increased available phosphorus by 169%, directly addressing the core issues of nitrogen and phosphorus deficiency in saline-alkali soils. Electrical conductivity also decreased by 17%, confirming that the strain enhanced nutrient availability by reducing stress. Hole application (T3) was less effective due to localized carrier enrichment, but still significantly superior to the control. This indicates that furrow application of the microbial agent has a greater impact on improvement efficiency.

[0074] Table 10 Effect of bacterial agent carrier on the growth promotion of crops in saline-alkali land

[0075] Index T1 (control) T2 (furrow application) T3 (hole application) Plant height (cm) 42.3±2.1 58.7±3.5* 54.2±2.8* Ear grain number (grains / ear) 24.5±1.8 38.2±2.4* 35.6±2.1* Yield (kg / ha) 2150±120 3250±180* 2980±150*

[0076] Note: Data are mean ± standard deviation (n=3), “*” indicates significant difference compared with the control (p<0.05).

[0077] Table 10 shows that the inoculant carrier significantly promoted crop growth by improving the rhizosphere environment. Furrow application (T2) and hole application (T3) increased wheat plant height by 38% and 28%, respectively, and grain number per ear by 56% and 45%, respectively. Ultimately, yield increased by 51% and 39% compared to the control. These results demonstrate that strain R-3's multiple growth-promoting mechanisms (nitrogen fixation, phosphate solubilization, and ACC deaminase) act synergistically in saline-alkali soils, significantly improving crop productivity.

[0078] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solutions obtained by equivalent replacement or equivalent transformation fall within the scope of protection of the present invention.

Claims

1. A Sphingomonas strain, characterized in that: The Sphingomonas ( Sphingomonas sp. ) was named R-3 and deposited in the General Microbiology Center of China Culture Collection Administration on May 30, 2025. The deposit number of the strain is: CGMCC No.34720.

2. A bacterial agent carrier produced using the Sphingomonas according to claim 1.

3. Use of the bacterial agent according to claim 1 or the bacterial agent carrier according to claim 2 in nitrogen fixation in saline-alkali land.

4. Use of the microbial agent according to claim 1 or the microbial agent carrier according to claim 2 in degrading organic phosphorus in saline-alkali land.

5. Use of the bacterial agent according to claim 1 or the bacterial agent carrier according to claim 2 in producing ACC deaminase.

6. A method for preparing the bacterial agent carrier according to claim 2, characterized in that: The specific steps include: S1. Fermentation broth treatment: Sphingomonas R-3 was inoculated into the culture medium and centrifuged to obtain the fermentation broth; S2. Encapsulation and granulation: the fermentation liquid is mixed with the sodium alginate solution, bentonite is added, cross-linked and solidified, and hot air drying is performed to obtain the bacterial agent carrier.

7. The method for preparing the microbial agent according to claim 6, characterized in that: In step S1, the culture medium formula is: 20 g / L glucose, 10 g / L tryptone, 1% NaCl, 0.3% L-tryptophan, pH 7.0; the culture conditions are: fermentation at 25-35°C and 160-200 rpm for 55-65 h; and the centrifugation conditions are: 7000-9000 rpm for 8-12 min.

8. The method for preparing the microbial agent according to claim 6, characterized in that: In step S2, the volume ratio of the fermentation liquid to the sodium alginate solution is 1:2-4.

Images