Sphingomonas, microbial inoculant, and preparation method and application thereof

Abstract

This invention belongs to the field of agricultural microbiology technology, and discloses a strain of Sphingomonas, a microbial agent, its preparation method and application, specifically disclosing a strain of Sphingomonas (… Sphingomonas Microbial agent (sp.) R7 was deposited on March 31, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025634. The invention discloses a microbial agent containing this bacterium, its preparation method, and its application in improving soil and / or promoting rice growth, as well as in reducing cadmium content in the aboveground parts of rice. This microbial agent not only increases the content of nutrients such as nitrogen and phosphorus in the soil, thus improving soil quality, but also significantly increases the dry matter weight of rice roots, stems, leaves, and brown rice, promoting rice plant growth; it also concentrates cadmium in the rice roots, reducing the cadmium content in the aboveground parts of rice.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, and particularly relates to a strain of Sphingomonas, microbial inoculants, their preparation methods and applications. Background Technology

[0002] Soil nutrient supply capacity is a crucial factor determining crop yield and quality. However, the low efficiency of traditional chemical fertilizer application has long been a problem. Studies show that only a small portion of nitrogen, phosphorus, and potassium fertilizers applied to the soil are absorbed and utilized by crops; the majority is lost through leaching, volatilization, or fixation, leading not only to resource waste but also environmental pollution. Currently, microbial inoculants are attracting attention due to their significant effects on nutrient transformation and crop growth promotion. However, traditional inoculants face a series of problems in field application, such as poor diffusion in the soil, making it difficult for active microorganisms to effectively cover the crop rhizosphere; low survival rate of strains, often rapidly inactivated due to changes in soil conditions (such as drought or high temperatures); and limited functionality, targeting only a single nutrient for decomposition and utilization. These problems significantly limit the role of microbial inoculants in improving nutrient utilization efficiency.

[0003] Soil cadmium pollution has become a major threat to food security. Cadmium is a highly toxic heavy metal that enters the soil through industrial emissions, fertilizers, and pesticides. Once in the soil system, cadmium is easily absorbed by plants and accumulates in crops, especially in food crops such as rice, posing a serious threat to human health through the food chain. Remediation methods for soil cadmium pollution mainly include chemical passivation and phytoremediation. However, chemical passivation agents are costly and may cause secondary pollution, while phytoremediation has a long cycle and limited effectiveness in remediating soils with high cadmium concentrations. Bioremediation technology has attracted attention due to its environmental friendliness and high efficiency; however, existing microbial strains used for remediating soil cadmium pollution exhibit limited adsorption capacity and poor environmental adaptability, hindering large-scale application. Especially in rice-growing environments, effectively passivating cadmium in the soil and reducing its accumulation in crops has become an urgent technical challenge. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide a strain of Sphingosine monocytogenes, a microbial inoculant, its preparation method and application.

[0005] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0006] A strain of Sphingosine monocytogenes ( Sphingomonas sp.), this sphingosomalid ( Sphingomonas sp.) was named Sphingosaminomonas ( Sphingomonassp.)R7 was deposited at the China Center for Type Culture Collection on March 31, 2025, with accession number CCTCC NO: M 2025634.

[0007] Based on a general inventive concept, the present invention also provides a microbial inoculant, comprising the above-mentioned Sphingomonas sphingosine monocytogenes (SMP). Sphingomonas sp.)R7.

[0008] Preferably, the microbial agent described above contains Sphingosine monocytogenes (SMP). Sphingomonas The concentration of sp.) R7 is (0.5-1.5)×10 8 cfu / mL. The strain concentration is further preferably (0.8-1.2) × 10⁻⁶. 8 cfu / mL, more preferably 1×10⁻⁶ 8 cfu / mL.

[0009] The aforementioned microbial agents, preferably, also include those for loading Sphingosine monocytogenes (SMP) (…). Sphingomonas A composite carrier for R7 sp., and for coating Sphingomonas (sp.) Sphingomonas The sustained-release material is of sp. R7; wherein the microbial agent has a microcapsule structure, the inner layer of which is loaded with sphingosine monocytogenes (sp.) R7. Sphingomonas The composite carrier of sp.)R7 has an outer layer of sustained-release material coating the composite carrier.

[0010] Preferably, the raw materials of the composite carrier in the above-mentioned microbial agent include rice husk ash, bentonite, and starch; and the raw materials of the slow-release material include sodium alginate, polyvinyl alcohol, and calcium chloride.

[0011] Based on a general inventive concept, the present invention also provides a method for preparing the above-mentioned microbial inoculant, comprising the following steps:

[0012] (1) Mix rice husk ash and bentonite, add water, then add starch, and then dry, crush and sterilize to obtain a composite carrier;

[0013] (2) Sphingosine monocytogenes ( Sphingomonas The bacterial suspension of sp.)R7 was mixed with the composite carrier obtained in step (1), and a protectant was added. After shaking and drying, a solid bacterial agent was obtained. The protectant was composed of sucrose, corn steep liquor powder and sodium chloride in a mass ratio of (3-7):3:1.

[0014] (3) The solid bacterial agent obtained in step (2) is added to a mixture of sodium alginate solution and polyvinyl alcohol aqueous solution, and then calcium chloride solution is added. After freeze drying, the microbial agent is obtained.

[0015] In the above preparation method, preferably, in step (1), the mass ratio of rice husk ash, bentonite and starch is (3-5):(2-4):(2-3); further preferably (3.5-4.5):(2.5-3.5):2, and even more preferably 4:3:2.

[0016] In the above preparation method, preferably, in step (1), the amount of water added is 4-6 times the total volume of rice husk ash and bentonite, more preferably 4.5-5.5 times, and even more preferably 5 times.

[0017] In the above preparation method, preferably, in step (1), ultrasonic treatment is performed after adding water, and the ultrasonic treatment time is 15-25 min, more preferably 18-22 min, and even more preferably 20 min.

[0018] In the above preparation method, preferably, in step (1), the drying method is oven drying, the drying temperature is 70-90℃, more preferably 75-85℃, and even more preferably 80℃; the drying time is 10-14h, more preferably 11-13h, and even more preferably 12h.

[0019] In the above preparation method, preferably, in step (1), the sterilization method is gamma ray sterilization, the radiation dose is 15-40kGy, more preferably 20-35kGy, and even more preferably 25kGy; the irradiation time is 20-26h, more preferably 22-25h, and even more preferably 24h.

[0020] In step (2), Sphingosomalidone (Sphingosomalidone) Sphingomonas The ratio of bacterial suspension to composite carrier of sp.)R7 is 1mL:(2-4)g, more preferably 1mL:(2.5-3.5)g, and even more preferably 1mL:3g.

[0021] Sphingosomalidone (Sphingosomalidone) Sphingomonas The mixing ratio of the bacterial suspension and the protectant of sp.)R7 is 1 mL: (7-11) g, more preferably 1 mL: (8-10) g, and even more preferably 1 mL: 9 g.

[0022] The Sphingosomalidone ( Sphingomonas The concentration of strain R7 in the bacterial suspension was (0.5-1.5)×10⁻⁶. 8 cfu / mL; the protective agent is composed of sucrose, corn steep liquor and sodium chloride in a mass ratio of (3-7):3:1, more preferably (4-6):3:1, and even more preferably 5:3:1.

[0023] In the above preparation method, preferably, in step (2), the oscillation speed is 150-200 rpm, more preferably 160-190 rpm, and even more preferably 180 rpm; the oscillation temperature is 25-30℃, more preferably 26-29℃, and even more preferably 28℃; the oscillation time is 1-3h, more preferably 1.5-2.5h, and even more preferably 2h.

[0024] In the above preparation method, preferably, in step (2), the drying method is spray drying, the temperature of the material inlet is 50-70℃, more preferably 55-65℃, and even more preferably 60℃; the temperature of the material outlet is 40-50℃, more preferably 42-48℃, and even more preferably 45℃; the drying time is 15-30s, more preferably 18-25s, and even more preferably 20s.

[0025] In step (3), the ratio of solid bacterial agent to mixed solution is 1g:(4-6)mL, more preferably 1g:(4.5-5.5)mL, and even more preferably 1g:5mL;

[0026] The volume ratio of sodium alginate solution to polyvinyl alcohol aqueous solution in the mixed solution is 1:(0.5-1.5), more preferably 1:(0.8-1.2), and even more preferably 1:1; the mass concentration of the sodium alginate solution is 12-18 g / L, more preferably 14-16 g / L, and even more preferably 15 g / L; the mass concentration of the polyvinyl alcohol aqueous solution is 2-5%, more preferably 3-4.5%, and even more preferably 4%.

[0027] This invention utilizes carrier and sustained-release microcapsule technology to load and encapsulate *Sphingomonas* strain R7. The carrier consists of rice husk ash, bentonite, and starch; the protective agents are sucrose, corn steep liquor, and sodium chloride; and the sterilization method is gamma ray sterilization. Rice husk ash, formed by high-temperature calcination, has a specific surface area of ​​50-80 m² / g. 2 / g, rich in surface hydroxyl groups (-OH), which can adsorb Cd 2+ It also provides sites for bacterial attachment; bentonite (montmorillonite) is a natural mineral raw material with low cost, and the cations in its crystal layers have exchangeability, which can fix Cd. 2+The above composite carriers form a triple cadmium passivation mechanism through physical adsorption of cadmium by rice husk ash, cadmium fixation through ion exchange of bentonite, and cadmium bioadsorption by bacteria. Furthermore, sucrose, a non-reducing sugar, acts as a protectant, stabilizing the bacterial cell membrane through hydrogen bonds. Corn steep liquor, rich in protein and amino acids, provides freeze-drying protection and slow-release nutrition during spray drying. Sodium chloride regulates osmotic pressure. Utilizing these protectants significantly reduces costs without affecting the survival rate of the bacterial strain. Gamma rays, with their strong penetrating and ionizing abilities, can penetrate the porous structure of composite carriers such as rice husk ash, bentonite, and starch, achieving uniform internal sterilization and avoiding sterilization dead zones caused by insufficient penetration in traditional high-temperature or chemical fumigation. Finally, sodium alginate and polyvinyl alcohol are used to protect the bacterial cells, improving the strain's tolerance during production and storage, and significantly enhancing survival rate and stability.

[0028] Based on a general inventive concept, the present invention also provides the above-mentioned Sphingosine Monoclonal bacteria ( Sphingomonas The application of microbial agents (spe.) or microbial agents prepared by the above-described preparation methods in soil improvement or rice growth promotion.

[0029] Based on a general inventive concept, the present invention also provides the above-mentioned Sphingosine Monoclonal bacteria ( Sphingomonas The application of microbial agents (spe.) or microbial agents prepared by the above-mentioned preparation methods in reducing the cadmium content in the above-ground parts of rice.

[0030] In the above-described application, preferably, during the three-leaf-one-heart stage of rice, in pot experiments, the microbial inoculant is applied at a ratio of 1:(80-120) of microbial inoculant to soil mass. A further preferred ratio of microbial inoculant to soil mass is 1:(90-110), and even more preferably 1:100.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] (1) The Sphingosine Monoclonal strain of the present invention ( Sphingomonas The R7 strain (sp.) possesses multiple biological functions, including phosphorus solubilization, nitrogen fixation, and cadmium tolerance. When prepared as a microbial agent, it can enhance soil fertility from the root, promote plant growth, and reduce the accumulation of heavy metal cadmium in the aboveground parts of rice. This achieves multiple goals of "improving soil, promoting growth, and ensuring safety," providing an efficient microbial solution for addressing issues such as soil infertility and heavy metal risks in agricultural production.

[0033] (2) The present invention designs the microbial agent into a double-layered slow-release structure of "composite carrier adsorption and fixation + gel microcapsule encapsulation". The inner layer is a composite carrier mainly composed of rice husk ash and bentonite, which provides a stable habitat and nutrient buffer for the bacteria. The outer layer is an interpenetrating gel network formed by sodium alginate-calcium and polyvinyl alcohol, which constitutes a slow-release capsule with physical barrier function. This structure realizes the controllable and slow release of bacteria in the soil, effectively solving the technical bottleneck of rapid inactivation and loss of bacteria due to environmental stress in traditional microbial agents, significantly prolonging the effective action time, and improving the stability and persistence of field application.

[0034] (3) The microbial agent prepared using the technical solution of this invention can not only better increase the content of nutrients such as nitrogen and phosphorus in the soil, thus improving the soil, but also significantly increase the dry matter weight of rice roots, stems, leaves, and brown rice, promoting the growth of rice plants; it can also enrich cadmium in the soil in the rice roots, reducing the cadmium content in the aboveground parts of rice. At the same time, the slow-release microcapsule technology used in this invention also realizes the gradual release of the strain in the soil, solving the problem that the strain is easily and rapidly inactivated or lost in the soil. This invention organically combines functional strains, inactivation carriers, and slow-release technology to form a high-performance agricultural microbial product that integrates "promoting growth, increasing fertilizer, reducing cadmium, and long-lasting effect", with significant ecological and economic benefits.

[0035] Biological Preservation Instructions

[0036] The seed endophytic bacterium involved in this invention is *Sphingosine monocytogenes*, with the Latin name... Sphingomonas sp., named Sphingosine Monoclonal ( Sphingomonas sp.)R7 was deposited at the China Center for Type Culture Collection on March 31, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025634. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 This is a phylogenetic tree constructed for the Sphingomonas R7 strain of the present invention;

[0039] Figure 2 A statistical graph showing the effect of different treatments on cadmium content in different parts of rice plants (different lowercase letters indicate significant differences between different treatments in the same part). P <0.05). Detailed Implementation

[0040] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0041] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0042] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0043] Example 1:

[0044] Isolation, screening and identification of Sphingosomalmonella R7:

[0045] Prepare the culture medium:

[0046] 1 / 2LB liquid culture medium: Weigh 5.0 g sodium chloride, 2.5 g yeast extract and 5.0 g tryptone, and bring the volume to 1000 mL with ultrapure water. Adjust the pH to 7.0, autoclave at 121°C for 30 minutes, and use after cooling.

[0047] NB liquid culture medium: Weigh 10.0 g of glucose, 3.0 g of beef extract and 5.0 g of peptone, and make up to 1000 mL with ultrapure water. Adjust the pH to 7.0, dispense into Erlenmeyer flasks, seal and autoclave at 121°C for 30 minutes. Use after cooling.

[0048] Phosphate-solubilizing bacteria screening medium: Weigh 5 g of tricalcium phosphate [Ca3(PO4)2], 10 g of glucose, 0.25 g of MgSO4·7H2O, 5 g of MgCl2·6H2O, 0.1 g of (NH4)2SO4, and 0.2 g of KCl. Make up to 1000 mL with ultrapure water, adjust the pH to 7.1~7.5, add 15 g of agar, and autoclave at 121℃ for 30 minutes. Cool to 50℃ and pour into plates.

[0049] Ashby nitrogen-fixing bacteria selection medium: Weigh 0.2 g KH2PO4, 10 g mannitol, 0.2 mL NaCl, 0.2 g MgSO4·7H2O, 5 g CaCO3, and 0.1 g CaSO4·2H2O, and bring the volume to 1000 mL with ultrapure water. Adjust the pH to 7.0, add 15 g agar, and autoclave at 121°C for 30 minutes. Cool to 50°C and pour into plates.

[0050] 1 / 2LB solid culture medium: Weigh 5.0 g sodium chloride, 2.5 g yeast extract and 5.0 g tryptone, and make up to 1000 mL with ultrapure water. Adjust the pH to 7.0, add 15 g agar, and autoclave at 121°C for 30 minutes. Cool to 50°C and pour into plates.

[0051] NB solid medium: Weigh 10.0 g glucose, 3.0 g beef extract and 5.0 g peptone, and bring the volume to 1000 mL with ultrapure water. Adjust the pH to 7.0, add 15 g agar, and autoclave at 121°C for 30 minutes. Cool to 50°C and pour into plates.

[0052] Select plump Huanghuazhan rice seeds from Zhouxi Town, Kaili City, Guizhou Province. First, soak the seeds in 75% anhydrous ethanol for 10 minutes, then pour off the ethanol and rinse five times with sterile water. Next, soak the seeds in 5% NaClO solution for 10 minutes, followed by five more rinses with sterile water. Spread 100 μL of the final rinse solution onto a 1 / 2 LB solid culture medium and incubate for 5 days. Observe for bacterial growth on the medium to ensure thorough sterilization of the rice seed surface.

[0053] After surface sterilization, rice seeds were placed in a sterile mortar, submerged in liquid nitrogen, and thoroughly ground into powder. Using a sterile weighing spoon, the rice seed powder was inoculated into 250 mL of 1 / 2 LB and NB liquid culture medium, respectively. The media were then incubated in a shaker (28℃, 180 rpm) for 36 hours. Afterward, 1 mL of the bacterial culture was diluted to 1×10⁻⁶. 4 1×10 5 and 1×10 6 After dilution, 100 μL of the diluted bacterial solution was spread onto NB solid medium and placed in an incubator (28°C) for inverted culture. The growth of the strains on the plates was observed periodically.

[0054] After the strains have grown for 5 days, use an inoculation loop to select strains with different morphological characteristics on the plate, streak them on the corresponding solid culture medium to obtain purified single bacteria. After activating the single bacteria, mix them with sterile glycerol in a ratio of 1:1, and aliquot 1.5 mL into 2 mL centrifuge tubes. Store them at -80℃ for later use.

[0055] The isolated endophytic bacteria were inoculated onto solid culture media used to test the phosphorus-solubilizing function of the strains. The presence of phosphorus-solubilizing zones on the plates was observed over 7 days. The presence of such zones indicated that the bacteria had phosphorus-solubilizing function.

[0056] The isolated endophytic bacteria were inoculated onto Ashby nitrogen-fixing bacteria selection medium, which is used to test the nitrogen-fixing function of the strains. Their growth was observed over 7 days, and growth indicated that the bacteria had nitrogen-fixing function.

[0057] The isolated endophytic bacteria were inoculated into 1 / 2 LB liquid medium and cultured in a shaker (28℃, 180 rpm) for 36 hours. 100 μL of the bacterial suspension was then spread onto 1 / 2 LB solid medium containing different Cd concentrations (1 mmol / L and 4 mmol / L) and placed in an incubator (28℃) for inverted culture. The growth was observed over 7 days, and growth indicated that the bacteria had cadmium tolerance.

[0058] The study found that one strain of bacteria simultaneously possesses phosphorus-solubilizing, nitrogen-fixing, and cadmium-tolerant functions. The physiological functions of this strain are shown in Table 1.

[0059] Table 1. Identification of physiological functions of the strains

[0060]

[0061] Note: "√" indicates that it has, "+" indicates that colonies have appeared, and "++" indicates that there are many colonies.

[0062] The isolated bacteria were subjected to 16S rDNA identification (by Shanghai Paisennong Biotechnology Co., Ltd.). The bacterial gene sequence was then compared online with the NCBI database, confirming that the isolated bacteria belonged to the [specific species not specified in the original text]. Sphingomonas (Sphingomonas genus). A phylogenetic tree for this strain was constructed using relevant software, such as... Figure 1 As shown, R7 falls on Sphingomonas Within the taxonomic group / branch formed by the reference sequence, multiple closely related sequences of the same genus cluster into the same lineage, further supporting its phylogenetic relationship. Sphingomonas It belongs to the bacteria.

[0063] The determined 16S rDNA sequence is shown in SEQ ID NO.1, and the specific sequence is as follows:

[0064]

[0065] The results showed that the bacteria isolated and screened above, which simultaneously possessed phosphorus-solubilizing, nitrogen-fixing, and cadmium-tolerant functions, were seed endophytic bacteria *Sphingosine monocytogenes*, with the Latin name *Sphingosine monocytogenes*. Sphingomonas sp., named Sphingosine Monoclonal ( Sphingomonas sp.)R7 was deposited at the China Center for Type Culture Collection on March 31, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025634.

[0066] Example 2:

[0067] This embodiment provides the preparation of microbial inoculants, including the following steps:

[0068] (1) Sphingosine monocytogenes ( Sphingomonas Preparation of R7 bacterial suspension (sp.)

[0069] The sphingosine monocytogenes obtained after isolation and purification from rice seeds in Example 1 ( Sphingomonas sp.) R7 was inoculated into 50 mL of LB liquid medium (pH 7.0) and activated for 36 h in a shaker (28℃, 180 rpm). Then, 2 mL of the activated bacterial solution was inoculated into 250 mL of LB liquid medium (pH 7.0) and cultured for 24 h in a shaker (28℃, 180 rpm). The fermentation broth was then transferred to a low-temperature high-speed centrifuge (4℃, 6000 rpm) and centrifuged for 10 min. The supernatant was removed, and the cells were resuspended in sterile ultrapure water. Cells were counted under an optical microscope using a cell counting chamber, and a 1×10⁻⁶ cell suspension was prepared. 8 CFU / mL bacterial suspension is available for use.

[0070] (2) Construction of solid microbial agents

[0071] Fresh rice straw was cleaned, cut into 3cm pieces, and calcined in a muffle furnace at 600℃ for 2 hours in an oxidizing atmosphere. After cooling, it was ground through a 100-mesh sieve to obtain rice husk ash (RHA). RHA was mixed with bentonite (BENT) and starch (ST) in a mass ratio of 4:3:2. First, the rice husk ash and bentonite were mixed in the specified ratio, then 5 times the volume of deionized water was added, and the mixture was ultrasonically dispersed for 20 minutes. Then, a 10% starch paste (prepared from the above-mentioned starch proportions) was added and stirred at 60℃ until a paste was formed. The paste was then spread evenly on a tray and dried in an oven at 80℃ for 12 hours. After pulverizing, it was passed through an 80-mesh sieve to obtain a composite carrier. The composite carrier was sterilized using gamma rays, with the radiation dose controlled within the range of 25 kGy. After irradiation for 24 hours, it was immediately sealed and stored in a dry, clean environment to prevent secondary contamination.

[0072] The sphingosine monocytogenes prepared in step (1) Sphingomonas The R7 bacterial suspension was mixed with the composite carrier at a ratio of 1:3 (mL / g), and then a protective agent consisting of 5% sucrose, 3% corn steep liquor and 1% sodium chloride was added (each percentage represents the mass-volume ratio of each component to the R7 suspension: g / 100mL). The mixture was shaken and adsorbed on a shaker (28℃, 180rpm) for 2 hours. Subsequently, a solid bacterial agent was obtained by spray drying. The inlet temperature of the material was controlled at 60℃ and the outlet temperature at 45℃ during the spray drying process, and the drying time of the material in the tower was 20s.

[0073] (3) Preparation of sustained-release microcapsules

[0074] Sodium alginate, a natural polymer, was selected to prepare a sodium alginate solution with a concentration of 15 g / L. Polyvinyl alcohol powder was weighed and dissolved in distilled water to prepare a 4% (w / w) polyvinyl alcohol aqueous solution. The sodium alginate solution and the polyvinyl alcohol aqueous solution were mixed in a 1:1 ratio, and the pH was adjusted to 7.0 to obtain a mixed solution, which was then sterilized for later use.

[0075] The solid bacterial agent prepared in step (2) was mixed with the sterilized mixed solution at a ratio of 1:5 (g / mL) and stirred evenly. At room temperature (25℃-30℃), a 2% sterilized calcium chloride (CaCl2) solution was added dropwise to allow the sodium alginate to react with the CaCl2 solution. 2 + A cross-linking reaction occurs, followed by gelation to form an encapsulation layer. The layer is then hardened for 10 minutes to ensure shell stability. The gelled microcapsules are placed in a freeze dryer pre-freezing chamber. The temperature is first lowered to -20°C at a rate of 10°C per half hour and held at this temperature for 2 hours. Then, the temperature is slowly lowered to -80°C at a rate of 5°C per minute and held for 2 hours. Subsequently, freeze-drying is performed under a vacuum of 20 Pa. The first drying rack temperature is -20°C for 10 hours, and the second drying rack temperature is 25°C for 4 hours to remove excess moisture and fix the morphology, yielding a microbial agent with a sustained-release microcapsule structure.

[0076] Example 3:

[0077] (a) Storage stability test

[0078] For step (1) of Example 2, preparation of Sphingosine Monoclonal (SMC) Sphingomonas A comparative storage stability test was conducted on the R7 bacterial suspension (treatment S) and the microbial inoculant prepared in Example 2 (treatment R). The initial viable counts of the two treatments were 1.0 × 10⁻⁶ and 1.0 × 10⁻⁶, respectively. 8 cfu / mL and 1.0×10 8 cfu / g.

[0079] The R7 bacterial suspension and microbial agent were separately aliquoted into aluminum foil bags of the same material, sealed, and stored at room temperature (25±2℃) under dry and light-protected conditions. Samples were taken at 0 and 1 month of storage. For treatment S, 1 mL of bacterial suspension was directly taken, serially diluted 10-fold, plated, incubated, and counted; the result is expressed as cfu / mL. For treatment R, 1.00 g of the sustained-release microcapsule microbial agent obtained in Example 2 was accurately weighed, added to 99 mL of sterile physiological saline, shaken for 30 min to allow the microbial agent to fully disintegrate and release the bacteria, and then serially diluted 10-fold and plated; the result is expressed as cfu / g. Treatments R and S were compared at the same dilution gradient.

[0080] The formula for strain survival rate is: Survival rate (%) = Number of viable bacteria at the end of storage / Number of viable bacteria at the beginning of storage × 100%.

[0081] Table 2 Comparison of storage stability of different treated microbial agents under normal temperature conditions

[0082]

[0083] Note: Different lowercase letters indicate significant differences between treatments. P <0.05).

[0084] The results are shown in Table 2. Under the same storage conditions, the viable cell counts of both the R7 bacterial suspension (treatment S) and the microbial agent (treatment R) decreased with prolonged storage time, but the rate of decrease differed significantly. After one month of storage, the survival rate of treatment S was only 38.98%, while the microbial agent with the sustained-release microcapsule structure maintained a survival rate of approximately 81.67%. This indicates that the microcapsule encapsulation structure can effectively slow down the activity decline of the R7 strain during storage, significantly improve the storage stability of the microbial agent, and is more suitable for commercial production and widespread application.

[0085] (ii) Impact on soil

[0086] A soil-cultured pot experiment was conducted in a greenhouse at the Hunan Provincial Rice Research Institute. Three treatment groups were set up: a blank control group (CK treatment), a group treated with R7 bacterial suspension (BS treatment), and a group treated with the microbial agent prepared in Example 2 (R7 treatment).

[0087] The rice variety was Huanghuazhan, purchased from Longping Seed Industry Direct Store in Changsha City, Hunan Province. After transplanting, at the three-leaf and one-heart stage, the rice was treated with BS (1 mL bacterial suspension: 100 g soil) and R7 (1 g microbial agent: 100 g soil), respectively, and applied to the soil around the rice roots. The control group (CK) was not treated with bacteria, but only had an equal amount of distilled water and soil added.

[0088] Soil and plant samples were collected at the rice maturity stage, and relevant indicators were measured. The results are shown in Table 3. Soil pH was determined using a potentiometric method at a soil-to-water ratio of 2.5:1 using a pH meter (Mettler-Toledo 320, Shanghai). Available phosphorus, nitrate nitrogen, and ammonium nitrogen in the soil were determined according to the methods described in "Soil Agricultural Chemical Analysis Methods" (Lu Rukun. Soil Agricultural Chemical Analysis Methods [M]. Beijing: China Agricultural Science and Technology Press, 2000).

[0089] Table 3 Effects of different treatments on soil physicochemical properties

[0090]

[0091] Note: Different lowercase letters indicate significant differences between treatments. P <0.05).

[0092] Compared with the control (CK) treatment, the R7 treatment significantly increased the available phosphorus and nitrate nitrogen content in the soil without significantly altering soil pH and ammonium nitrogen levels, demonstrating stronger nutrient activation and inorganic nitrogen supply capacity. Available phosphorus content was significantly higher than that of the CK treatment, showing the most prominent improvement. Nitrate nitrogen exhibited a significant gradient of "R7>BS>CK," indicating that R7 has a clear advantage in improving soil phosphorus availability and promoting the conversion of mineral nitrogen to available forms.

[0093] (III) Impact on rice

[0094] A soil-cultured pot experiment was conducted in a greenhouse at the Hunan Provincial Rice Research Institute. Three treatment groups were set up: a blank control group (CK treatment), a group treated with R7 bacterial suspension (BS treatment), and a group treated with the microbial agent prepared in Example 2 (R7 treatment).

[0095] Rice seedlings were transplanted to cadmium-contaminated soil at the three-leaf stage (total cadmium content was 1.31 mg / kg, and available cadmium content was 0.86 mg / kg). One week after the rice seedlings began to recover, the seedlings were treated with BS (1 mL of bacterial suspension: 100 g of soil) and R7 (1 g of microbial agent: 100 g of soil), respectively, and applied to the soil around the rice roots. The control group (CK) was not treated with microbial agents, but only had an equal amount of distilled water and soil added.

[0096] Rice plants were sampled at maturity, and the dry matter weight and cadmium content of the roots, stems, leaves, and brown rice were determined. The results are shown in Table 4. The rice plants were digested using a diluted nitric acid microwave digestion method (see IJSDS A, AFL A, APSP B, et al. Microwave-assisted digestion employing diluted nitricacid for mineral determination in rice by ICP OES [J]. Food Chemistry, 2020, 319(2):126435.). The cadmium content was then detected using inductively coupled plasma mass spectrometry (ThermoScientific™ iCAP™ PRO XP ICP-OES).

[0097] Table 4. Effects of different treatments on the dry matter weight of mature rice plants.

[0098]

[0099] Note: Different lowercase letters indicate significant differences between treatments. P <0.05).

[0100] The results showed that, compared with the control group (CK) without bacterial treatment, the group treated with the microbial agent prepared in Example 2 significantly increased the dry matter weight of rice roots, stems, leaves, and brown rice (Table 4), with increases of 20.99%, 30.49%, 21.51%, and 12.76%, respectively. This further verifies that the application of the microbial agent prepared in this invention is beneficial to the growth of rice plants.

[0101] At the rice maturity stage, the cadmium content in different parts of the rice plant in all three treatments was in the following order: roots > stem sheath > leaves > brown rice (e.g., ...). Figure 2 (As shown). Compared with the control (CK), the cadmium content in rice plants treated with the microbial agent prepared in Example 2 (R7 treatment) showed a significant decreasing trend in rice stems, leaves, and brown rice, but the opposite trend was observed in the roots, with an increase of 29.20% compared to the CK. The R7 treatment was similar to the BS treatment in leaves and brown rice, but higher than the BS treatment in roots and stems. This indicates that the R7 treatment is more effective in enhancing cadmium retention in roots and stems, and overall reduces the cadmium risk in brown rice.

[0102] Example 4:

[0103] (I) Effects of different proportions of composite carrier on the survival rate of bacterial strains and available phosphorus content in soil

[0104] Based on the preparation process of the microbial agent in Example 2, two additional control groups were set up, with only the composition ratio of the composite carrier changed, to explore the effects of different mass ratios on the survival rate of Sphingosomalidone R7 in the microbial agent and the effect on improving available phosphorus in the soil.

[0105] Treatment Group A: namely, the microbial inoculant in Example 2.

[0106] Treatment Group B: The preparation process of the microbial agent is the same as in Example 2, except that the mass ratio of rice husk ash: bentonite: starch is replaced with 3:3:3.

[0107] Treatment Group C: The preparation process of the microbial agent is the same as in Example 2, except that the mass ratio of rice husk ash: bentonite: starch is replaced with 5:2:2.

[0108] The survival rate of the bacterial strains was determined using the plate count method (immediately after the corresponding group of bacterial agents was prepared). Survival rate = (number of viable bacteria after spray drying / number of viable bacteria before spray drying) × 100%.

[0109] At the three-leaf and one-heart stage of rice, the microbial agent was applied to the soil of Huang Huazhan rice pots at a weight ratio of 1:100. Soil samples were collected at maturity, and the available phosphorus content in the soil was detected by the molybdenum antimony colorimetric method (the basic available phosphorus content was 25.6 mg / kg).

[0110] Table 5. Effects of different carrier ratios on the survival rate of bacterial strains and available phosphorus content in soil.

[0111]

[0112] Note: Different lowercase letters indicate significant differences between treatments. P <0.05).

[0113] The results are shown in Table 5. The survival rate of the strain in treatment A was 82.42%, significantly higher than that in treatments B (65.17%) and C (71.59%). Furthermore, the available phosphorus content in the soil was the highest at 31.45 mg / kg, significantly higher than the baseline available phosphorus content. This demonstrates that the carrier ratio specified in Example 2 of this application can optimally protect the activity of the strain and efficiently enhance soil nutrients.

[0114] (II) Effects of different sucrose addition amounts on the survival rate of R7 strain after spray drying

[0115] Based on the preparation process of the microbial agent in Example 2, two additional control groups were set up, with only the amount of sucrose added being changed, to investigate the effect of different amounts of sucrose added on the survival rate of R7 strain after spray drying.

[0116] Treatment Group D: The preparation process of the microbial agent is the same as in Example 2, except that the amount of sucrose added in the protectant is replaced with 3%.

[0117] Treatment Group E: This refers to the preparation process of the microbial inoculant in Example 2.

[0118] Treatment group F: The preparation process of the microbial agent is the same as in Example 2, except that the amount of sucrose added in the protectant is replaced with 7%.

[0119] The survival rate of the strains after spray drying in each group was calculated using the plate count method.

[0120] Table 6. Effects of different sucrose addition amounts on the survival rate of R7 strain after spray drying.

[0121]

[0122] Note: Different lowercase letters indicate significant differences between treatments. P <0.05).

[0123] The results are shown in Table 6. The strain with 5% sucrose addition in treatment E had the highest survival rate, which avoided damage to the bacteria during spray drying and is the preferred sucrose addition amount of the present invention.

[0124] The above results indicate that by using the preferred composite carrier ratio (4:3:2) and protectant ratio (5% sucrose addition) of this invention, a microbial agent with the highest strain survival rate and the best effect on improving available phosphorus in the soil can be prepared. This provides a material basis and quality assurance for the significant application effects of the agent (such as growth promotion and cadmium reduction) in subsequent embodiments.

[0125] Example 5:

[0126] Based on the data results from Example 3, three soil concentrations of cadmium pollution—low (0.3 mg / kg), medium (1 mg / kg), and high (2 mg / kg)—were established. At the three-leaf stage of rice, the microbial inoculant was applied to the potted rice soil of Huang Huazhan at a weight ratio of 1:100 (microbial inoculant prepared in Example 2: soil). Rice plant samples were collected at maturity to test the cadmium content of brown rice and the dry matter weight of roots. A control group (CK) was also established, where the microbial inoculant prepared in Example 2 was not applied during the three-leaf stage.

[0127] Table 7. Application effects of microbial agents in soils with different cadmium contamination concentrations.

[0128]

[0129] Note: Different lowercase letters indicate significant differences between treatments. P <0.05).

[0130] The results are shown in Table 7. In low, medium, and high cadmium-contaminated soils, the microbial agent prepared in Example 2 significantly reduced the cadmium content in brown rice and increased the dry matter weight of roots. In the medium-low cadmium soils, the cadmium content in brown rice decreased to below 0.20 mg / kg (meeting the GB 2762-2022 cadmium limit of 0.2 mg / kg for rice), demonstrating that the agent has excellent cadmium passivation and growth-promoting effects under different cadmium pollution scenarios, and has a wide range of applications.

[0131] In summary, the *Sphingomonas sphingosine monocytogenes* R7, an endophytic bacterium isolated and purified from rice seeds in this invention, possesses phosphorus-solubilizing, nitrogen-fixing, and cadmium-tolerant functions. The microbial agent prepared by double-layer encapsulating the R7 strain using a carrier and slow-release microcapsule technology improves the R7 strain's tolerance during production and storage, significantly enhancing its survival rate and stability. Furthermore, it enables the gradual release of *Sphingomonas sphingosine monocytogenes* R7 strain into the soil, solving the problem of rapid inactivation or loss of the strain in soil. This allows the microbial agent to better enhance soil fertility, reduce cadmium content in the aboveground parts of rice, and promote rice growth and development.

[0132] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of Sphingosine monocytogenes (SSM-1) Sphingomonas sp.), characterized in that, The sphingomonas strain was named Sphingomonas R7 and was deposited at the China Center for Type Culture Collection (CCTCC) on March 31, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025634.

2. A microbial inoculant, characterized in that, Including Sphingomonas R7 as described in claim 1.

3. The microbial agent as described in claim 2, characterized in that, The concentration of Sphingomonas R7 in the microbial inoculant is (0.5-1.5) × 10⁻⁶. 8 cfu / mL.

4. The microbial agent as described in claim 2, characterized in that, It also includes a composite carrier for loading Sphingomonas R7 and a sustained-release material for coating Sphingomonas R7; wherein the microbial agent has a microcapsule structure, with the inner layer being the composite carrier loading Sphingomonas R7 and the outer layer being the sustained-release material coating the composite carrier.

5. The microbial agent as described in claim 4, characterized in that, The raw materials for the composite carrier include rice husk ash, bentonite, and starch; the raw materials for the slow-release material include sodium alginate, polyvinyl alcohol, and calcium chloride.

6. A method for preparing a microbial inoculant as described in any one of claims 2 to 5, characterized in that, Includes the following steps: (1) Mix rice husk ash and bentonite, add water, then add starch, and then dry, crush and sterilize to obtain a composite carrier; (2) Mix the bacterial suspension of Sphingomonas R7 with the composite carrier obtained in step (1), add a protectant, shake and dry to obtain a solid bacterial agent; the protectant is composed of sucrose, corn steep liquor powder and sodium chloride in a mass ratio of (3-7):3:

1. (3) The solid bacterial agent obtained in step (2) is added to a mixture of sodium alginate solution and polyvinyl alcohol aqueous solution, and then calcium chloride solution is added. After freeze drying, the microbial agent is obtained.

7. The preparation method according to claim 6, characterized in that, In step (1), the mass ratio of rice husk ash, bentonite and starch is (3-5):(2-4):(2-3). In step (2), the ratio of Sphingomonas R7 bacterial suspension to the composite carrier is 1 mL:(2-4) g; the ratio of Sphingomonas R7 bacterial suspension to the protectant is 1 mL:(7-11) g; and the concentration of the strain in the Sphingomonas R7 bacterial suspension is (0.5-1.5) × 10⁻⁶. 8 cfu / mL; In step (3), the ratio of solid bacterial agent to mixed solution is 1g:(4-6)mL; the volume ratio of sodium alginate solution and polyvinyl alcohol aqueous solution in the mixed solution is 1:(0.5-1.5), the mass concentration of sodium alginate solution is 12-18g / L, and the mass concentration of polyvinyl alcohol aqueous solution is 2-5%.

8. The use of a sphingomonas strain as described in claim 1, or a microbial agent as described in any one of claims 2-5, or a microbial agent prepared by any one of claims 6-7, in improving soil and / or promoting rice growth.

9. The application of a sphingomonas strain as described in claim 1, or a microbial agent as described in any one of claims 2-5, or a microbial agent prepared by any one of claims 6-7, in reducing the cadmium content of brown rice.

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