Sphingomonas sp. with cellulose degradation, growth promotion and alleviation of plant nacl stress and application thereof

Abstract

The application discloses a sphingomonas capable of degrading cellulose, promoting growth and relieving NaCl stress of plants and an application thereof. The application relates to the field of microorganisms, and provides the sphingomonas, or offspring thereof, which is sphingomonas indicans (Sphingomonas indicans) Sphingomonas rhizoryzae ) with a strain number of RS6 and a registration number of CGMCC No. 31493 in the General Microbiological Center of China Microorganism Strain Preservation Management Committee. The strain RS6 provided by the application is a new strain of sphingomonas, and has the capability of degrading cellulose. Potted plant tests show that, compared with a non-inoculation control, after the strain is inoculated under NaCl stress, the aboveground fresh weight and the underground fresh weight of rice can be increased, the strain can be used as a microbial organic fertilizer to improve soil fertility, relieve NaCl stress of crops and improve the adaptability of crops to NaCl stress.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a sphingosine monocytogenes strain that degrades cellulose, promotes growth, and alleviates NaCl stress in plants, and its applications. Background Technology

[0002] The rhizosphere microbiome is the microbial community surrounding plant roots, which is crucial for plant growth and health. It can improve soil nutrient utilization and help plants resist biotic stresses such as pathogens and pests, as well as abiotic stresses such as drought and salinity.

[0003] Numerous PGPR groups have been discovered both domestically and internationally. These microorganisms possess multiple functions, including secreting plant hormones, dissolving soil phosphorus, releasing iron, and decomposing potassium. Among them, *Sphingomonas* spp. ( Sphingomonas Members of this genus are widely distributed in nature, and strains of this genus possess multiple functions, such as producing indole-3-acetic acid (IAA), generating siderophores, and dissolving phosphates. Furthermore, they have been reported in areas such as pollutant degradation and environmental remediation. Summary of the Invention

[0004] The purpose of this invention is to provide a sphingomonas strain that can degrade cellulose, promote growth, and alleviate NaCl stress in plants, and its applications.

[0005] In a first aspect, the present invention claims protection for a sphingomonas bacterium or its descendants.

[0006] The *Sphingospora* species claimed in this invention is *Sphingospora riceensis* (… Sphingomonas rhizomycetes Its strain number is RS6, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 31493.

[0007] Rice rhizosporium ( Sphingomonas rhizoryzae RS6 is a Gram-negative bacterium. After 3 days of growth on TSA solid medium, the colonies are yellow, round, raised, with smooth edges, viscous, and glossy, with a colony diameter of 1-2 mm. This strain has excellent plant growth-promoting properties and can significantly alleviate the stress of NaCl on plants.

[0008] Secondly, the present invention claims protection for compositions containing Sphingomonas or its progeny as described in the first aspect above.

[0009] Further, the composition may be a culture, which is a substance obtained by culturing the Sphingomonas or its progeny described in the first aspect above in a microbial culture medium (all substances in the culture container, i.e., fermentation products, such as fermentation broth containing the Sphingomonas or its progeny and substances secreted into the liquid culture medium, or solid fermentation product containing the Sphingomonas or its progeny and substances secreted into the solid culture medium).

[0010] The microbial culture medium may be a bacterial culture medium. The bacterial culture medium may be a solid culture medium or a liquid culture medium.

[0011] In the above-mentioned cultures, the substances include the sphingomonas or its progeny (the bacterial cells themselves) and / or its metabolites described in the first aspect above.

[0012] The term "metabolite" refers to the primary and / or secondary metabolites produced during microbial metabolism. Primary metabolism refers to the process by which microorganisms absorb various nutrients from the external environment and, through catabolism and anabolism, generate substances and energy to sustain life activities. The products of primary metabolism are called primary metabolites, such as monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids, and various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, and lipids. Secondary metabolism refers to the process by which microorganisms, at a certain growth stage, use primary metabolites as precursors to synthesize substances that have no clearly defined function for their life activities. The products of secondary metabolism are called secondary metabolites, and are mostly compounds with relatively complex molecular structures. Based on their functions, they can be classified into types such as antibiotics, hormones, alkaloids, and toxins.

[0013] The term "culture" refers to any liquid or solid culture medium that has grown a microbial community after artificial inoculation and cultivation. It is the product obtained by growing and / or amplifying microorganisms; it can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites, or other components produced during the cultivation process. The term "culture" also includes passaged cultures obtained by subculturing microorganisms; these can be cultures of a single generation or mixtures of several generations.

[0014] Furthermore, the composition may be a microbial agent, a microecological preparation, or a bio-fertilizer.

[0015] In the aforementioned microbial agents, microecological preparations, or biofertilizers, the active ingredients may be *Sphingomonas* or its progeny, metabolites of *Sphingomonas* or its progeny, and / or cultures of *Sphingomonas* or its progeny, as described in the first aspect above. The active ingredients may also contain other biological and / or non-biological components. Those skilled in the art can determine the other active ingredients of the microbial agents, microecological preparations, or biofertilizers based on the desired effects.

[0016] In addition to the active ingredients, the aforementioned microbial agents, microecological preparations, or biofertilizers may also contain a carrier. The carrier may be a biologically inert carrier commonly used in the pesticide field. The carrier may be a solid carrier or a liquid carrier; the solid carrier may be a mineral material, plant material, or polymer compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material may be at least one of wheat flour, soybean flour, and starch; the polymer compound may be polyvinyl alcohol and / or polyethylene glycol; the liquid carrier may be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent may be decane and / or dodecane.

[0017] In the above-mentioned microbial agents, microecological preparations, or bio-fertilizers, the dosage form of the microbial agents, microecological preparations, or bio-fertilizers can be various, such as liquid, emulsion, suspension, powder, granules, wettable powder, or water-dispersible granules.

[0018] Depending on the requirements, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc. may also be added to the microbial agents, microecological preparations, or bio-fertilizers.

[0019] In the bacterial agent, microecological preparation, or bio-fertilizer, the sphingomonas or its progeny and / or the metabolites of the sphingomonas or its progeny may be present in the form of cultured live cells, fermentation broth of live cells, filtrate of cell culture, or a mixture of cells and filtrate.

[0020] In this document, the metabolites of *Sphingomonas* or its progeny can be obtained from the fermentation broth of *Sphingomonas* or its progeny. The metabolites of *Sphingomonas* or its progeny can be sterile metabolites of *Sphingomonas* or its progeny or bacterial metabolites of *Sphingomonas* or its progeny. Specifically, the sterile metabolites of *Sphingomonas* or its progeny (sterile fermentation filtrate) can be prepared as follows: *Sphingomonas* or its progeny are cultured in a liquid culture medium, and the *Sphingomonas* or its progeny are removed from the liquid culture (fermentation broth) by filtration, thus obtaining the sterile metabolites of *Sphingomonas* or its progeny. Specifically, the bacterial metabolites of *Sphingomonas* or its progeny can be prepared as follows: *Sphingomonas* or its progeny are cultured in a liquid fermentation medium, and the fermentation broth—containing *Sphingomonas* or its progeny and substances secreted into the liquid culture medium—is collected; this fermentation broth is the bacterial metabolites of *Sphingomonas* or its progeny.

[0021] Furthermore, the composition has at least one of the following properties:

[0022] A1) Degradation of cellulose;

[0023] A2) Relieve NaCl stress in plants;

[0024] A3) Enhances POD activity in plants;

[0025] A4) Reduce the MDA content in plants;

[0026] A5) Promotes plant growth;

[0027] A6) Promotes an increase in the underground fresh weight of plants;

[0028] A7) Promotes an increase in the aboveground fresh weight of plants.

[0029] Thirdly, the present invention claims the use of *Sphingomonas* or its descendants as described in the first aspect above, or the composition described in the second aspect above, wherein the use is any of the following:

[0030] B1) Applications in the degradation of cellulose or in the preparation of products for the degradation of cellulose;

[0031] B2) Applications in alleviating NaCl stress in plants or in the preparation of products for alleviating NaCl stress in plants;

[0032] B3) Applications in enhancing the POD activity of plants or in the preparation of products for enhancing the POD activity of plants;

[0033] B4) Application in reducing the MDA content of plants or in the preparation of products for reducing the MDA content of plants;

[0034] B5) Application in promoting plant growth or in the preparation of products for promoting plant growth;

[0035] B6) Application in promoting the increase of underground fresh weight of plants or in the preparation of products for promoting the increase of underground fresh weight of plants;

[0036] B7) Application in promoting the increase of aboveground fresh weight of plants or in the preparation of products for promoting the increase of aboveground fresh weight of plants.

[0037] Furthermore, the improvement of plant POD activity can be achieved by enhancing plant POD activity under NaCl stress conditions.

[0038] Furthermore, the reduction of plant MDA content can be achieved by increasing plant MDA content under NaCl stress conditions.

[0039] Furthermore, the promotion of plant growth can be achieved by promoting plant growth under NaCl stress conditions.

[0040] Furthermore, the promotion of increased underground fresh weight of plants can be achieved by promoting increased underground fresh weight of plants under NaCl stress conditions.

[0041] Furthermore, the promotion of aboveground fresh weight increase can be achieved under NaCl stress conditions.

[0042] Fourthly, the present invention claims a method for alleviating NaCl stress in plants.

[0043] The method for alleviating NaCl stress in plants claimed by this invention may include the following steps: treating the plant to be treated or its growth substrate with the Sphingomonas bacillus or its progeny described in the first aspect above or the composition described in the second aspect above, thereby alleviating the NaCl stress in the plant.

[0044] In some embodiments of the present invention, the treatment involves drenching the plant with a bacterial suspension containing the sphingomonas or its progeny.

[0045] Fifthly, the present invention claims a method for promoting plant growth.

[0046] The method for promoting plant growth claimed in this invention may include the following steps: treating the plant to be treated or its growth substrate with the Sphingomonas bacillus or its progeny as described in the first aspect above or the composition as described in the second aspect above, thereby promoting the growth of the plant.

[0047] Furthermore, the promotion of plant growth can be achieved by promoting plant growth under NaCl stress conditions.

[0048] In some embodiments of the present invention, the treatment involves drenching the plant with a bacterial suspension containing the sphingomonas or its progeny.

[0049] Sixthly, the present invention claims a method for culturing Sphingomonas or its progeny as described in the first aspect above.

[0050] The method for culturing Sphingomonas or its progeny as described in the first aspect of the present invention includes the step of culturing Sphingomonas or its progeny in a culture medium for culturing microorganisms.

[0051] In a seventh aspect, the present invention claims a method for preparing a composition.

[0052] The method for preparing the composition claimed in this invention includes the step of using Sphingomonas or its progeny as a component of the composition as described in the first aspect above; the composition is the composition described in the second aspect above.

[0053] In some embodiments of the present invention, the NaCl stress is simulated using a NaCl solution with a final concentration of 120 mmol / L.

[0054] In all the aforementioned relevant aspects, the plant may be any of the following:

[0055] C1) Angiosperms;

[0056] C2) Monocotyledons;

[0057] C3) Plants of the order Poales;

[0058] C4) Gramineae plants;

[0059] C5) Plants of the genus *Oryza*;

[0060] C6) Rice.

[0061] Experiments have shown that the *Sphingomonas oryzae* strain provided in this invention (…) Sphingomonas rhizoryzae RS6 is a new species of *Sphingosaminomonas*. *Sphingosaminomonas rice* ( Sphingomonas rhizoryzae RS6 has the ability to degrade cellulose. Pot experiments showed that, compared with the uninoculated negative control group, inoculation with *Sphingomonas oryzae* (rhizoma oryzae) significantly reduced cellulose content. Sphingomonas rhizoryzae RS6 can increase both the above-ground and below-ground fresh weight of rice. *Sphingosine monocytogenes* (rice root sphingosine monocytogenes) Sphingomonas rhizoryzae RS6 can be used as a microbial organic fertilizer to improve soil fertility, alleviate NaCl stress in crops, and enhance crop adaptability to NaCl stress.

[0062] Preservation Instructions

[0063] Classification and nomenclature: *Sphingosine monocytogenes* (rice root sphingosine mono Sphingomonas rhizoryzae );

[0064] Biological material from ginseng: RS6;

[0065] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0066] The abbreviation for the depository institution is CGMCC.

[0067] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0068] Deposit date: July 30, 2024;

[0069] Registered with the China National Collection Center (CGMCC) No. 31493. Attached Figure Description

[0070] Figure 1 Rice rhizosporium (Sphingomonas oryzae) Sphingomonas rhizoryzae The colony morphology of RS6 after 3 days of incubation on TSA plates.

[0071] Figure 2 To construct *Sphingosaminomonas rice* using the neighbor-joining method based on the 16S rRNA gene sequence ( Sphingomonas rhizoryzae Phylogenetic tree of RS6 and related model bacteria. Note: The numbers in parentheses are the GenBank sequence numbers of the strain's 16S rRNA gene sequence; the reference strains in the figure are all model strains of their respective species. Novosphingobiuum post-mortem L3E4 T (KT337427) is an outgroup.

[0072] Figure 3 To construct a strain containing rice rhizosphingosphytosphingosimus (rhizosphingosphytosphingosimus) using the maximum likelihood method, based on 92 core bacterial genes tandemly. Sphingomonas rhizoryzae RS6 and Sphingomonas Phylogenetic tree of closely related species within the genus.

[0073] Figure 4 Rice rhizosporium (Sphingomonas oryzae) Sphingomonas rhizoryzae ) RS6 test results on cellulose degradation ability.

[0074] Figure 5 The growth status of rice seedlings inoculated with strain RS6 and uninoculated with strain RS6 under NaCl stress at 27 days. The left side shows the seedlings inoculated with strain RS6, and the right side shows the seedlings uninoculated.

[0075] Figure 6 The figures show the aboveground and underground fresh weights of rice seedlings under 27 days of NaCl stress. The *** in the figure indicates... P <0.01.

[0076] Figure 7 This figure shows the determination of the environmental stress response and antioxidant capacity of rice seedlings under 27 days of NaCl stress. In the figure, ** and *** represent... P <0.01. Detailed Implementation

[0077] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0078] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0079] The formulations of the various culture media and solutions involved in the following examples are as follows:

[0080] (1) TSB liquid culture medium: 15.0g tryptone, 5.0g soybean peptone, 5.0g sodium chloride, 1000mL distilled water, adjust pH to 7.3±0.2, sterilize at 121℃ for 15min.

[0081] (2) TSA solid medium (TSA plate): Add 20.0g agar to TSB liquid medium and sterilize at 121℃ for 15min.

[0082] (3) Starch medium: NA medium, 0.2% (w / v) soluble starch, pH 7.4, 121℃, sterilized for 20 min.

[0083] (4) Casein culture medium: Solution a: 5g skim milk powder, 50mL distilled water; Solution b: 50mL NB, 1.5g agar. Sterilize solutions a and b at 121℃ for 15min, and after cooling to about 60℃, mix well and dispense into plates.

[0084] (5) Cellulose Congo Red Medium:

[0085] Sodium nitrate 1g, dimethyl hydrogen phosphate 1.2g, potassium dihydrogen phosphate 0.9g, magnesium sulfate 0.5g, potassium chloride 0.5g, yeast extract powder 0.5g, acid-hydrolyzed casein 0.5g, Congo red 0.2g, cellulose powder 5.0g, agar 20g, distilled water 1000ml. Sterilize at 121℃ for 15 minutes.

[0086] Example 1: Sphingosine monocytogenes of rice ( Sphingomonas rhizoryzae Separation and identification of RS6

[0087] I. Sphingosine monocytogenes of rice ( Sphingomonas rhizoryzae RS6 separation

[0088] Rice rhizosphere soil samples were collected in Kenli District, Dongying City, Shandong Province (118.58°E, 37.58°N). The samples were transported back to the laboratory and stored at 4°C using an ice box. Intact rice roots were taken, and excess soil was removed using sterile forceps. The roots were placed in Erlenmeyer flasks containing sterile water and shaken at 150 rpm for 30 minutes at room temperature. Root tissue was then picked out with forceps, centrifuged, and the supernatant was discarded; the precipitate was the rhizosphere soil. 1 g of rhizosphere soil was weighed and resuspended in 10 mL of sterile water for serial dilution. 100 μL of each dilution was spread onto TSA plates and incubated upside down at 30°C for one week. Based on physiological morphology, single colonies were picked using a bamboo stick and inoculated onto plates for purification. After confirming pure cultures, the cultures were transferred to slant culture for short-term storage at 4°C, and then transferred to 20% glycerol tubes for long-term storage at -80°C. One of the isolated and purified strains was named RS6.

[0089] II. *Sphingosine monocytogenes* (rice root sphingosine monocytogenes) Sphingomonas rhizoryzae RS6 identification

[0090] 1. Morphological identification of strains

[0091] The RS6 strain, isolated and purified in step one above and in the logarithmic growth phase with stable colony size, was described as a single colony, including colony size, color, transparency, colony surface condition, and colony edge condition. Following the manufacturer's instructions, a Gram staining kit from Solarbio Technology Co., Ltd. (Beijing) was used to Gram stain the RS6 smears, and the morphology of the bacteria was observed using an optical microscope.

[0092] The colonies of strain RS6 on TSA plates are yellow, round, raised, with smooth edges, viscous, and glossy, with a colony diameter of 1-2 mm. Figure 1 The cells are Gram-negative, rod-shaped, and do not form spores.

[0093] 2. Molecular identification

[0094] Following the instructions, genomic DNA was extracted using the TIANamp bacterial genomic DNA extraction kit from Beijing Tiangen Biotech Co., Ltd. 16S rRNA gene amplification was performed using universal bacterial primers 27F (SEQ ID NO:1, 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO:2, 5'-GGTTACCTTGTTACGACTT-3'). The 50 μL PCR amplification system consisted of: 25 μL 2×Taq PCR Mix, 2 μL 27F (10 μmol / L), 2 μL 1492R (10 μmol / L), 19 μL ddH2O, and 2 μL DNA template. The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 56℃ annealing for 1 min, 72℃ extension for 90 s, 30 cycles; and a final extension at 72℃ for 10 min. After the PCR amplification products were verified by 1% agarose gel electrophoresis, the positive PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequenced sequences were uploaded to Ezbiocloud (www.ezbiocloud.net / eztaxon) for sequence alignment.

[0095] The sequencing length of the 16S rRNA gene of strain RS6 is 1484 bp (SEQ ID No. 3). Comparison results from the EzBioCloud database show that strain RS6 is similar to... Sphingomonas pokkalii L3B27 T (98.65%) Sphingomonas kyeonggiensis THG-DT81 T (98.34%) Sphingomonas pituitosa EDIV T (98.15%) and Sphingomonas azotifigens NBRC 15497 T (98.01%) of the sequences showed high similarity and were all below the classification threshold (98.8%) for describing the species. 16S rRNA gene sequences highly similar to strain RS6 were retrieved from the EzBioCloud server and aligned using MUSCLE. A phylogenetic tree was constructed using the Neighbour-Joining method with MEGA X software. The evolutionary distance using the NJ method was calculated using the Kimura two-parameter model, with a bootstrap value of 1000. The phylogenetic tree constructed using the Neighbour-Joining method is shown below. Figure 2 As shown, strain RS6 and strain Sphingomonas pokkalii L3B27 T Clustered together, yet forming a separate branch, this indicates that strain RS6 is... Sphingomonas A potential new species of the genus.

[0096] 3. Genome analysis

[0097] Genomic DNA was sent to Annoroad Gene Technology (Beijing) Co., Ltd., where a draft genome sequence of strain RS6 was performed using an Illumina NovaSeq 6000 sequencing system. Assembly was performed using SPAdes software, yielding 35 contigs, N 50 The length is 414781 bp. The genome size is 3.67 Mb, and the G+C content is 66.51%. The ANIm method in the pyANI software was used to analyze strain RS6 and... Sphingomonas Genome-wide average nucleotide identity (ANI) analysis of closely related strains. The digital DNA-DNA hybridization (dDDH) values ​​between strain RS6 and the reference strain were compared using the Genome-to-Genome Distance Calculator (GGDC) 3.0 server (https: / / ggdc.dsmz.de / ggdc.php#).

[0098] Strain RS6 and others with publicly available genome sequences Sphingomonas Compared to the type strains of the same genus, the ANI values ​​were 73.71-82.60%, lower than the previously proposed critical value of 95-96% for species delimitation; the dDDH values ​​of strain RS6 and its type strain were between 19.20-26.30%, far below the 70% species delimitation threshold. See Table 1 for details. Both the ANI and dDDH results indicate that strain RS6 is a... Sphingomonas A new species of the genus.

[0099]

[0100] To further clarify the taxonomic position of strain RS6, this invention performed a genomic phylogenetic analysis, using UBCG software to classify strain RS6 and... Sphingomonas Thirty-two type strains of the genus were analyzed, and 92 orthologous genes were tandemly analyzed. A gene sequence phylogenetic tree was constructed using the maximum likelihood method. Figure 3 The Bootstrap value was 1000. The results showed that strain RS6 and strain... Sphingomonas pokkalii L3B27 T (GCA_003096275.1) and Sphingomonas Several strains of the genus clustered together and formed a separate branch, indicating that strain RS6 is... Sphingomonas A potential new species of the genus.

[0101] 4. Physiological and chemical classification and identification

[0102] Add several drops of 5% H2O2 to a glass petri dish, pick strain RS6 and react with it. If bubbles are produced, it proves that the strain can produce catalase. Spot strain RS6 on filter paper soaked in 1% p-aminoxylamine hydrochloride, using Pseudomonas aeruginosa and Escherichia coli as positive and negative controls, respectively. If a rose-red ring appears around the colony, it indicates that it can produce catalase. Spot strain RS6 on five points on starch medium, setting up three replicates, and incubate at 25°C for 2-5 days. After removing the plates, add iodine solution around the colonies and observe the color change. If a colorless transparent ring appears around the colony, it indicates that the bacteria have produced amylase and diffused into the substrate, hydrolyzing the starch in the medium into a substance that does not react with iodine; if the area around the colony is blue, it indicates that the bacteria do not produce amylase. Five spots of strain RS6 were inoculated onto casein medium, with three replicates. The plates were incubated at 25°C for 7 days. After removing the plates, the casein around and below the colonies was observed to see if it was decomposed into clear zones. If it was clear, it indicated that the strain had the ability to hydrolyze casein.

[0103] The results showed that strain RS6 produced bubbles after contact with 5% H2O2, indicating that its catalase result was positive; strain RS6 did not produce a color change after contact with filter paper moistened with 1% p-aminoxylamine hydrochloride, indicating that its oxidase result was negative; strain RS6 did not produce a clear zone on either casein medium or starch medium with added iodine solution, indicating that strain RS6 does not have the ability to hydrolyze casein and starch.

[0104] The enzyme activity and carbohydrate utilization of strain RS6 and related model strains were determined using API 20NE, ZYM, and 50 CH test strips (bioMérieux) from bioMérieux, France.

[0105] The 20NE test results showed that strain RS6 exhibited positive results in the hydrolysis reaction of aesculin and the presence of p-nitro- β The D-galactoside hydrolysis reaction was positive, while the nitrate reduction reaction, indole reaction, acidification reaction, arginine hydrolysis reaction, urease hydrolysis reaction, and gelatin hydrolysis reaction were negative; it can assimilate mannose, N Acetyl-glucosamine, maltose, and malic acid cannot assimilate glucose, arabinose, mannitol, gluconate, decanoic acid, adipic acid, citric acid, and phenylacetic acid.

[0106] In the ZYM enzyme activity identification assay, alkaline phosphatase, esterase (C4), lipoesterase (C8), valine aromatic aminoaminase, leucine aromatic aminoaminase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, β -Galactosidase, β - Glucuronidase, β- Glucosidase, result positive; Lipoase (C14), Cystine Aromatase, Trypsin, Chymotrypsin, α -Galactosidase, α -glucosidase, N -acetyl-glucosaminease, α -Mannosidase, α - Fucosidase result was negative;

[0107] Results from 50 CH showed that strain RS6 could hydrolyze L-arabinose, D-xylose, D-galactose, D-glucose, D-mannose, L-rhamnose, and methyl- α -D-glucopyranoside, N - Acetylglucosamine, aesculin, salicin, D-lactose, D-cellobiose, D-maltose, D-mercaptobiose, D-sucrose, D-trehalose, D-maltotriose, D-raffinose, starch, D-gentiobiose, D-torunose, D-lysose, and D-fucose; cannot be hydrolyzed mannitol, erythritol, D-arabinose, D-ribose, L-xylose, D-calendol I, methyl- β- D-xylopyranoside, L-sorbose, eurythritol, inositol, mannitol, sorbitol, methyl- α -D-Mannopyranoside, amygdalin, inulin, glycogen, xylitol, L-fucose, D-arabinol, L-arabinol, potassium gluconate and 2-keto-gluconate; weakly utilized D-fructose, D-tagatose and 5-keto-gluconate.

[0108] The physiological and biochemical characteristics of strain RS6 differ from those of related model strains, as shown in Table 2.

[0109]

[0110] Note: + indicates positive or usable; - indicates negative or unusable; w indicates weak positive.

[0111] Based on the above identification, it can be confirmed that the strain RS6 obtained in this invention is *Sphingosine Monoclonalella* (SCM). Sphingomonas A new bacterial species has been named *Sphingosaminomonas rice* (…). Sphingomonas rhizoryzae The strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 30, 2024, with the registration number CGMCC No. 31493. It will be referred to as strain RS6 below.

[0112] Example 2, *Sphingomonas oryzae* (rice root sphingosine monocytogenes) Sphingomonas rhizoryzae ) Detection of RS6's ability to degrade cellulose

[0113] Sphingosine monocytogenes (Synthia sphingosine monocytogenes) Sphingomonas rhizoryzaeInoculate RS6 onto cellulose Congo red medium and incubate at 30°C for 3 days. Observe whether there is a light red transparent ring around the colony. If there is, it indicates that the colony can secrete cellulase.

[0114] The results showed that after 3 days of growth on cellulose Congo red medium and subsequent staining, *Sphingosine monocytogenes* (rice root sphingosine monocytogenes)... Sphingomonas rhizoryzae RS6 can produce a pale red transparent zone; this indicates that *Sphingomonas oryzae* (rhizoma oryzae) can produce a transparent zone. Sphingomonas rhizoryzae RS6 has the ability to degrade cellulose. Figure 4 ).

[0115] Example 3, *Sphingomonas oryzae* (rice root sphingosine monocytogenes) Sphingomonas rhizoryzae RS6 alleviates NaCl stress in plants

[0116] Select rice rhizosporium (Sphingomonas oryzae) Sphingomonas rhizoryzae A single RS6 colony was inoculated into a 500 mL Erlenmeyer flask containing 200 mL of TSB liquid medium and incubated at 30 °C for 48 h. The colony was then centrifuged at 8000 rpm for 10 min, and the cells were collected and resuspended in TSB liquid medium to obtain an OD value. 600 =1.0 bacterial suspension (with uninoculated TSB liquid medium as the control group for seed soaking). Uniformly sized rice varieties (SE362) were selected (described in "R. Quan, J. Wang, H. Qin, L. Chen, D. Xiao, Z. Zhao, Z. Zhang, X. Zhu, Z. Li, R. Huang, Improving grain yield and salttolerance by optimizing plant height with beneficial haplotypes in rice"). Rice ), J. Adv. Res. (2024) Seeds (Table S1) from the supplementary materials of article S2090123224005630) were sown in 32-cell seedling trays containing 3713g of a mixture of vermiculite and nutrient soil (vermiculite: nutrient soil = 1:1), with 6 seeds per cell. An RS6 inoculation experimental group (labeled YB119) and a control group (CK, labeled Y119) were established, each with 8 cells. After most of the rice seeds germinated, thinning was performed, leaving 4 rice seedlings of similar growth per cell. When the seedlings reached the one-leaf-one-heart stage (approximately 7 days), NaCl stress treatment was applied by irrigating each tray with 8L of salt solution (final NaCl concentration 120mmol / L). 24 hours after salt treatment, a bacterial suspension was applied to the roots of the rice seedlings in the experimental group, with 0.5mL of the bacterial suspension per seedling. The control group was irrigated with uninoculated pure TSB liquid medium. Watering was performed every 3 days during the plant growth period. The experiment was conducted in a greenhouse.

[0117] Figure 5 The growth status of rice seedlings at 27 days. Under NaCl stress, rice seedlings exhibited symptoms such as leaf drying, curling, and shedding; plant height was significantly reduced; and the overall plant showed a wilting trend. Different treatments resulted in varying degrees of salt damage. Agronomic traits of rice seedlings were also observed. Figure 6 As shown, the aboveground and underground fresh weights of rice seedlings in the experimental group were 0.4280±0.0278g and 0.4310±0.1279g, respectively, while those in the control group were 0.3560±0.0401g and 0.3280±0.0512g, respectively. Both the aboveground and underground fresh weights of the experimental group were significantly higher than those of the control group. P <0.01). This indicates inoculation with *Sphingomonas oryzae* (rhizoma oryzae) (…). Sphingomonas rhizoryzae Treatment with RS6 can promote the growth of rice seedlings under NaCl stress.

[0118] To further investigate the ability of strain RS6 to alleviate NaCl stress in rice, the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), malondialdehyde (MDA), and reduced glutathione (GSH) in the leaves were measured. Appropriate amounts of rice seedling leaves were cut into 2.0 mL centrifuge tubes, quickly placed in liquid nitrogen, and ground into powder. SOD, CAT, POD, MDA, and GSH in the leaves of rice seedlings from both the experimental and control groups were extracted and measured according to the instructions provided in the respective kits: Superoxide Dismutase (SOD) Activity Kit (Beijing Sangon Biotech, China), Catalase (CAT) Activity Kit (Beijing Sangon Biotech, China), Peroxidase (POD) Activity Kit (Beijing Sangon Biotech, China), Malondialdehyde (MDA) Content Kit (Beijing Sangon Biotech, China), and Reduced Glutathione (GSH) Content Kit (Beijing Sangon Biotech, China).

[0119] The results showed no significant differences in catalase (CAT) activity, superoxide dismutase (SOD) activity, and reduced glutathione (GSH) content between the experimental group (RS6) and the control group (CK). The POD activity in the experimental group was significantly higher than that in the control group. Figure 7 , P <0.01), NaCl stress leads to excessive accumulation of reactive oxygen species (such as H2O2) in plants, which can damage cell membrane structure and function. POD catalyzes the production of H2O and O2 from H2O2. POD plays an important role in the reactive oxygen species scavenging system, and higher POD indicates that the plant has strong antioxidant capacity and resistance to stress. The above results indicate that *Sphingosphomonas rice* (… Sphingomonas rhizoryzaeRS6 can stimulate plants to produce highly active POD, enabling them to better cope with NaCl stress and promoting plant growth; at the same time, the MDA content in the experimental group was significantly lower than that in the control group. Figure 7 , P <0.01), reactive oxygen species (ROS) are metabolic products in plants. Stress exacerbates the production of large amounts of ROS. ROS attacks unsaturated fatty acids in the cell membrane, triggering lipid peroxidation and producing MDA. The accumulation of MDA further damages the structure and function of the cell membrane, leading to increased cell membrane permeability, leakage of intracellular substances, and affecting the normal physiological activities of the plant. In summary, it is speculated that *Sphingosphomonas rice* (… Sphingomonas rhizoryzae RS6 alleviates NaCl stress in rice seedlings and improves rice's NaCl stress tolerance by inducing plants to produce large amounts of POD and reducing MDA accumulation.

[0120] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Sphingosine monocytogenes, characterized in that: The sphingosomal bacillus is *Sphingosomalobacterium oryzae* (rhizome sphingosomalbacterium). Sphingomonas rhizoryzae Its strain number is RS6, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 31493.

2. The use of the Sphingomonas strain of claim 1 in the degradation of cellulose or in the preparation of products for the degradation of cellulose.

3. The use of the Sphingomonas strain of claim 1 in alleviating NaCl stress in plants or in the preparation of products for alleviating NaCl stress in plants; The method of alleviating NaCl stress in plants is to increase the POD activity of plants, reduce the MDA content of plants, promote the increase of underground fresh weight and / or promote the increase of aboveground fresh weight of plants under NaCl stress conditions. The plant in question is rice.

4. The use of the Sphingomonas strain of claim 1 in promoting plant growth or in the preparation of products for promoting plant growth; The promotion of plant growth refers to promoting the increase of underground fresh weight and / or the increase of aboveground fresh weight of plants under NaCl stress conditions. The plant in question is rice.

5. A method for alleviating NaCl stress in plants, comprising the following steps: treating the plant to be treated with the Sphingomonas strain of claim 1, thereby alleviating the NaCl stress in the plant; The method of alleviating NaCl stress in plants is to increase the POD activity of plants, reduce the MDA content of plants, promote the increase of underground fresh weight and / or promote the increase of aboveground fresh weight of plants under NaCl stress conditions. The plant in question is rice.

6. A method for promoting plant growth, comprising the following steps: treating the plant to be treated with the Sphingomonas strain of claim 1, thereby promoting the growth of the plant; The promotion of plant growth refers to promoting the increase of underground fresh weight and / or the increase of aboveground fresh weight of plants under NaCl stress conditions. The plant in question is rice.

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