Rice endophytic sphingosine vector SY02 and application thereof

Abstract

The invention belongs to the technical field of agricultural microorganisms, and particularly relates to rice endophytic sphingosine vector SY02 and application thereof. The preservation number of the rice endophytic sphingobium yanoikuyae SY02 provided by the invention is CCTCC (China Center For Type Culture Collection) NO: M2026182, and the sphingobium yanoikuyae SY02 has the functions of producing indoleacetic acid, promoting plant growth and seed germination, promoting adventitious bud proliferation of tissue culture seedlings and relieving browning. The sphingosine vector bacterium and the microbial agent prepared through strain fermentation have the advantages of being green, safe, efficient, long-acting and the like, and have good application prospects in the aspects of promoting plant growth and seedling breeding.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a rice endophytic sphingosphate bacterium SY02 and its application. Background Technology

[0002] With the development of agricultural modernization, chemical fertilizers and growth regulators are widely used in agricultural production, significantly improving crop yields and tissue culture seedling propagation efficiency for a certain period. However, long-term excessive use of chemical agents can easily lead to problems such as soil compaction, decreased soil fertility, and environmental pollution. It may also result in harmful substance residues in agricultural products, threatening human health.

[0003] Traditional tissue culture seedling proliferation relies on plant hormones (6-BA, NAA, 2,4-D, etc.), mineral nutrients, and carbon sources in chemical culture media. However, this hormone dependence leads to weak seedling quality and low proliferation coefficients. Plant endophytes are microbial communities that live within plants and form a mutually beneficial symbiotic relationship with the host plant. Some endophytes can secrete plant growth hormones (such as auxins and cytokinins), dissolve insoluble minerals in the soil, and inhibit the growth of plant pathogens, thereby promoting the growth and development of the host plant and improving its stress resistance. Compared with chemical agents, using endophytes as tissue culture seedling proliferation promoters has advantages such as being environmentally friendly, sustainable, and residue-free, meeting the needs of green agricultural development.

[0004] Existing technologies indicate that beneficial bacteria promote proliferation from three dimensions: endogenous regulation, nutrient supply, and microenvironment optimization, with a gentler and more sustainable effect. Endophytic bacteria mainly regulate the differentiation and proliferation of tissue culture seedlings by secreting endogenous substances such as auxin (IAA), cytokinin (CTK), and gibberellin (GA3), synthesizing and secreting growth-promoting substances homologous to plants to replace or assist the effects of chemical hormones, avoiding the side effects of high-concentration chemical hormones. Alternatively, they can alleviate oxidative stress in tissue culture seedlings by inhibiting contamination from other microorganisms, such as browning and apoptosis caused by ROS accumulation; or they can secrete antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), while synthesizing antioxidant substances such as flavonoids and phenols to clear ROS in tissue culture seedlings, alleviate browning and oxidative stress, and maintain normal cell division and proliferation.

[0005] Yano Sphingosine mononitrate ( Sphingobium yanoikuyae *Sphingosporium yanoense* is a Gram-negative bacterium belonging to the genus *Sphingosporium* in the family Sphingomonasceae. Existing research indicates that this bacterium has potential in the degradation of environmental pollutants. Some literature reports that *Sphingosporium yanoense* induces root IAA synthesis, promoting root development and biomass accumulation. However, no reports have been found regarding its role as a plant endophyte in promoting flavonoid synthesis, seed germination and crop growth, tissue culture seedling proliferation, or preventing browning in tissue culture seedlings.

[0006] Therefore, screening a plant endophytic sphingosine bacterium and developing its applications is of great significance for promoting crop growth and seedling propagation. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a strain of *Sphingosine monocytogenes* and its applications. This strain is an endophytic bacterium found in rice and has significant effects on promoting plant growth and tissue culture seedling proliferation. It can be applied in agricultural production and plant tissue culture.

[0008] An endophytic *Sphingosine monocytogenes* strain SY02 in rice, wherein *Sphingosine monocytogenes* ( Sphingobium yanoikuyae The accession number of SY02 is CCTCC NO: M 2026182, the accession date is January 22, 2026, and it is deposited at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China.

[0009] This invention protects a biological agent comprising a suspension of *Sphingosphobiella yanoides* SY02 as described in claim 1 and / or a fermentation broth of *Sphingosphobiella yanoides* SY02.

[0010] Furthermore, the biological agent has at least one of the following functions: (1) It produces indoleacetic acid, which promotes plant growth and seed germination; (2) Promote the proliferation of adventitious buds in tissue culture seedlings and alleviate browning of tissue culture seedlings.

[0011] This invention also protects the application of the rice endophytic sphingomyelin-2 or the bacterial agent in promoting plant growth, seed germination, promoting the proliferation of adventitious buds in tissue culture seedlings, and preventing browning of tissue culture seedlings.

[0012] Furthermore, the OD of the bacterial suspension of the rice endophytic *Sphingosine monocytogenes*... 600 The range is 0.1 to 1.33.

[0013] Furthermore, the method for preparing the fermentation broth of the rice endophytic sphingosine monocytogenes includes the following steps: S1 used an inoculation loop to scrape the strain cultured for 7-10 days from PDA, inoculated it into an Erlenmeyer flask containing 200 mL of liquid PDA, and placed it on a shaker at 200 RPM and 28℃ for 24 h to obtain the seed culture of Yano sphingosine strain SY02. S2 was cultured by adding 1 mL of the seed culture obtained from S1 to Erlenmeyer flasks containing 200 mL of PDA and R2A media respectively. The culture time in PDA media was 4–26 h, and the culture time in R2A media was 4–14 h.

[0014] Furthermore, the PDA culture medium is prepared by boiling 200 g of peeled potatoes in 1000 mL of water for 15 min, filtering the potato residue through four layers of gauze, adding 20 g of glucose, adding water to bring the volume to 1000 mL, and autoclaving at 121°C for 25 min. The R2A culture medium consisted of: 0.25 g / L tryptone, 0.5 g / L acid-hydrolyzed casein, 0.5 g / L soluble starch, 0.1 g / L magnesium sulfate, 0.25 g / L peptone, 0.5 g / L yeast extract, 0.5 g / L glucose, 0.3 g / L dipotassium hydrogen phosphate, and 0.3 g / L sodium pyruvate, and was autoclaved at 121°C for 25 min.

[0015] Compared with the prior art, the present invention has the following beneficial effects: Experiments have shown that the rice endophytic sphingosine monocytogenes (Sphingosine monocytogenes) provided by this invention ( Sphingobium yanoikuyae SY02, which produces indoleacetic acid, significantly promotes plant growth and seed germination, significantly promotes the proliferation of adventitious buds in tissue culture seedlings, and alleviates browning in tissue culture seedlings. The *Sphingosine monocytogenes* and the microbial agent prepared by fermentation of the strain provided by this invention have advantages such as being green, safe, efficient, and long-lasting, and have good application prospects in plant growth and seedling propagation. Attached Figure Description

[0016] Figure 1 The growth of rice propagation seedlings under different conditions for 60 days is shown. Figure 1 a represents normally cultured rice propagation seedlings; Figure 1 b represents rice seedlings contaminated with bacteria SY01; Figure 1 c represents rice seedlings contaminated with other bacteria; Figure 2 Phylogenetic tree of bacteria SY01 and SY02; Figure 3 Scanning electron microscope image of *Sphingosine monocytogenes* (SY02); Figure 4 The strain characteristics of *Sphingosine monocytogenes* (SY02) are as follows: 4a shows the nitrogen fixation function of strain SY02; 4b shows the phosphorus solubilization (organic phosphorus) function of strain SY02; 4c shows the phosphorus solubilization (inorganic phosphorus) function of strain SY02; 4d shows the amylase production function of strain SY02; 4e shows the siderophore production function of strain SY02; and 4f shows the IAA production function of strain SY02. Figure 5 The colony growth of *Sphingosine monocytogenes* (SY02) on three different solid culture media is shown. Figure 5 a, Figure 5 b、 Figure 5c shows the colony growth on rice propagation subculture medium, PDA medium, and R2A medium, respectively. Figure 6 The growth curves of *Sphingosine monocytogenes* (SY02) in different liquid culture media are shown. Figure 6 a represents the growth curve of SY02 in PDA liquid culture; Figure 6 b is the growth curve of SY02 in R2A liquid medium; Figure 6 c represents the growth curve of SY02 in rice propagation liquid medium; Figure 6 d is a comparison graph of the growth of SY02 in three liquid culture media; Figure 7 Germination images of rice varieties LTH and ZH11 on day 3; Figure 8 The effects of different dilutions of SY02 fermentation broth on seed germination and growth of rice variety ZH11 on day 24. Figure 9 This diagram illustrates the effect of *Sphingomonas yannoides* (SY02) on the proliferation of tissue-cultured shoots in different crops. Figure 9 a, Figure 9 b、 Figure 9 c shows adventitious buds on day 5 of subculture of aloe vera, strawberry, and cardamom. Figure 9 d、 Figure 9 e Figure 9 f shows the adventitious buds of aloe vera, strawberry, and cardamom when cultured without SY02. Figure 9 g、 Figure 9 h、 Figure 9 i represents the adventitious buds of aloe vera, strawberry, and cardamom when SY02 was added for cultivation; Figure 10 A graph showing the variation in diameter of browned roots in aloe seedlings treated with *Sphingosine monocytogenes* (SY02). Figure 10 and Figure 10 b is a comparison graph showing the changes in the browning diameter of the roots of aloe vera seedlings in the SY02 treatment and control groups. Figure 11 GO enrichment analysis of differentially expressed transcripts of aloe vera treated with SY02; Figure 12 This is a KEGG enrichment analysis diagram of SY02-induced aloe vera ascending transcripts. Detailed Implementation

[0017] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions.

[0018] Example 1: Discovery, isolation, culture and identification of the strain This invention provides an endophytic strain of *Sphingosine monocytogenes* in rice (… Sphingobium yanoikuyae It was found in rice tissue culture bottles during the sixth generation of subculture of rice tissue culture.

[0019] 1. Rice tissue culture methods S1. Explant selection: Select mature, plump, and disease-free high-quality rice seeds.

[0020] S2. Sterilization of explants: After peeling the selected seeds, soak them in 0.1% streptomycin for 15 min, then soak them in 0.1% HgCl2 for 5 min, and finally rinse them twice with sterile water.

[0021] S3. Adventitious bud differentiation culture: Sterilized rice seeds were inoculated into seed germination medium for culture. Seeds germinated after 3-4 days, and cultured for another 10-15 days to obtain seedlings 5-6 cm in height. Culture conditions: Temperature: 28±2℃; Light duration: 10 h / d; Light intensity: 1600-2000 Lx. The seed germination medium was: MS 4.4 g / L + 6-BA 0.2 mg / L + NAA 0.2 mg / L + agar 5.0 g / L + sucrose 30 g / L, pH 5.8-6.0.

[0022] S4. Proliferation (subculture) culture: Seedlings are inoculated into proliferation medium. Culture conditions: temperature: 28±2℃; light duration: 10 h / d; light intensity: 1600-2000 Lx. Culture for 20-25 days to obtain uncontaminated clump-forming seedlings, achieving bud proliferation. The proliferation (subculture) medium is: MS 4.4 g / L + 6-BA 1.2-1.5 mg / L + NAA 0.1-0.2 mg / L + agar 5.0 g / L + sucrose 30 g / L, pH 5.8-6.0.

[0023] 2. Discovery of the strain When rice was subcultured to the sixth generation, a bacterium was found on the culture medium of the previously uncontaminated tissue culture seedlings. However, the growth of the tissue culture seedlings was not inhibited, and after further subculture, the differentiation of adventitious buds was more vigorous than that of the uncontaminated seedlings. Figure 1 b). Under normal conditions, bacterial contamination of tissue culture seedlings will inhibit growth or even lead to death. Figure 1 c), therefore, it is believed that this bacterium can promote the proliferation of adventitious shoots in rice, and this strain is predicted to be an endophytic microorganism in rice. Figure 1 ).

[0024] 3. Isolation, culture, and identification of bacteria on culture medium in tissue culture flasks

[0025] 4. Isolation and identification of bacteria in rice plants Leaves of rice tissue culture seedlings infected with this strain and exhibiting vigorous bud differentiation were removed in a clean bench, washed 10 times with sterile water, and then ground. The ground solution was spread on rice proliferation medium and incubated at 30 ℃. After bacterial growth, the high concentration of bacteria was diluted and cultured. Regular observation was conducted, and single colonies with different morphologies were selected for purification. Through repeated streak plating and purification, rice endophytic microbial strains were obtained. These strains were compared with bacteria on the culture medium in the tissue culture flasks. Strains with similar colony morphology were selected, and Sanger bidirectional sequencing was performed using universal primers 16S-27F and 16S-1492R, yielding a 1348 bp sequence, which, upon alignment, was identical to that shown in SEQ ID NO.1.

[0026] The gene sequences obtained from sequencing were compared with those in the NCBI database and compared with those of *Sphingosine monocytogenes* (Yano). Sphingobium yanoikuyae With 100% similarity, a phylogenetic tree was constructed to confirm taxonomic status. Figure 2 At the molecular level, the bacterium was confirmed to be *Sphingosine monocytogenes*. S. yanoikuyae The strain isolated from the tissue culture bottle was named SY01, and the strain isolated from the rice plant was named SY02.

[0027] 5. Morphological identification of *Sphingosine monocytogenes* SY02 Sphingosine monocytogenes SY02 forms round, yellow, opaque colonies on the surface of rice propagation medium. The surface is smooth, moist, and mucous-like, with regular edges. Under a scanning electron microscope and an optical microscope, the bacterium appears as rod-shaped. Figure 3 ), consistent with the characteristics of sphingosine bacteria.

[0028] Example 2: Strain characteristics of *Sphingosine monocytogenes* SY02 The SY02 strain was tested using five methods: nitrogen fixation capacity, phosphorus solubilization capacity, amylase production capacity, siderophore production capacity, and IAA production capacity. The specific methods are as follows: 1. Nitrogen fixation (1) Preparation of Ashby nitrogen-free solid culture medium (1 L): 10 g glucose, 0.2 g 0.02% K2HPO4, 0.2 g sodium chloride, 0.2 g magnesium sulfate monohydrate, 0.2 g potassium sulfate, 5 g calcium carbonate, 15-20 g agar, 1 L distilled water, sterilized at 121℃ for 20 min.

[0029] (2) Inoculate strain SY02 onto Ashby nitrogen-free solid medium. The experiment included 6 biological replicates. After incubation at 30°C for 3 days, observe the colonies. If there is a clear transparent zone of dissolution around the colonies, it proves that the strain has nitrogen-fixing ability.

[0030] (3) Detection results: No transparent dissolution zone was formed around the colony of strain SY02 ( Figure 4 a) This experiment proves that this strain does not have the ability to fix nitrogen.

[0031] 2. Phosphorus solubilization (1) Preparation of organic phosphorus and inorganic phosphorus solid culture media: Weigh 13.16 g of organic phosphorus bacterial culture medium or 32.0 g of inorganic phosphorus bacterial culture medium, respectively, heat and dissolve in 1000 mL of distilled water, and autoclave at 121℃ for 15 min to obtain organic phosphorus solid culture medium and inorganic phosphorus solid culture medium.

[0032] Organic phosphorus solid culture medium composition (g / L): glucose 10.0, ammonium sulfate 0.5, yeast extract 0.5, sodium chloride 0.3, potassium chloride 0.3, magnesium sulfate 0.3, ferrous sulfate 0.03, manganese sulfate 0.03, lecithin 0.2, calcium carbonate 1.0, pH value: 7.0-7.5 (25℃).

[0033] Inorganic phosphorus solid culture medium composition (g / L): glucose 10.0, ammonium sulfate 0.5, yeast extract 0.5, sodium chloride 0.3, potassium chloride 0.3, magnesium sulfate 0.3, ferrous sulfate 0.03, manganese sulfate 0.03, calcium phosphate 5.0, agar 15.0, pH 7.0-7.5 (25℃).

[0034] (2) The strain SY02 was inoculated onto organic and inorganic phosphorus solid culture media respectively, and observed after incubation at 30°C for 3 days. The experiment included 6 biological replicates. If a transparent lysis zone formed around the colony, it proved that the strain had phosphorus solubilizing ability.

[0035] (3) Detection results: On organic and inorganic phosphorus solid culture media, no transparent dissolution zone was formed around the colonies of strain SY02 ( Figure 4 b, Figure 4 c), This experiment proves that this strain does not have the ability to solubilize phosphorus.

[0036] 3. Production of amylase (1) Preparation of PDA culture medium and 5% Lugol's iodine solution PDA medium preparation: Boil 200 g of peeled potatoes in 1000 mL of water for 15 min, filter the potato residue through four layers of gauze, add 20 g of glucose and 20 g of agar powder, add water to make up to 1000 mL, and autoclave at 121℃ for 25 min.

[0037] Preparation of Lugo's iodine solution (5%): Take about 50 mL of distilled water, add 10 g of potassium iodide, stir thoroughly until completely dissolved, gradually add 5 g of elemental iodine to the above solution, continue stirring until completely dissolved, then dilute to 100 mL with distilled water and mix well, and store in a brown reagent bottle.

[0038] (2) Inoculate strain SY02 onto PDA solid medium and incubate at 30°C for 2 days. Add one drop of Lugol's iodine solution to each single colony that grows. Repeat the experiment 6 times. If a transparent zone of dissolution forms around the colony, it proves that the strain has the ability to dissolve amylase.

[0039] (3) Detection results: No transparent dissolution zone was formed around the colony of strain SY02 ( Figure 4 d), This experiment proves that this strain does not have the ability to produce amylase.

[0040] 4. Iron-producing carrier (1) Preparation of CAS detection medium: Take 10.87 g of the medium and dissolve it in 1 L of deionized water. Heat and boil for more than 1 min to dissolve it. After dispensing, autoclave at 115℃ for 20 min.

[0041] CAS assay medium composition (g / L): Chromium azurite S (CAS) 60.5 mg, hexadecyltrimethylammonium bromide (HDTMA) 72.9 mg, FeCl3·6H2O 2.645 mg, agar 9000 mg, 0.1 mol / L phosphate buffer 50 mL (containing NaH2PO4·2H2O 295.25 mg, NaH2PO4·12H2O 1213.5 mg, ammonium chloride 125 mg, potassium dihydrogen phosphate 37.5 mg, sodium chloride 62.5 mg), pH: 6.8±0.2 (25 ℃).

[0042] (2) Inoculate strain SY02 onto CAS detection medium and incubate at 30℃ for 5 days, then observe the colonies. Repeat the experiment 6 times. If there is a clear transparent zone of dissolution around the colonies, it proves that the strain has the ability to produce siderophores.

[0043] (3) Detection results: No obvious transparent dissolution zone was found around the colony of strain SY02. Figure 4 e), This experiment demonstrates that this strain does not possess the ability to produce siderophores.

[0044] 5. Production of IAA (1) King's B liquid culture medium formula (1 L): 20 g peptone, 15 mL glycerol, 1.5 g K2HPO4, 0.1 g tryptophan, 1 L distilled water, pH 7.2±0.2 (20 min). Salkowski's colorimetric reagent: Slowly add 150 mL concentrated sulfuric acid to 250 mL deionized water while stirring. After the solution cools, add 7.5 mL of 0.5 mol / L FeCl3·6H2O solution.

[0045] (2) Strain SY02 was inoculated onto King'B liquid medium and cultured at 180 RPM / min and 28℃ for 3 days. 500 μL of each culture was centrifuged, and the supernatant was mixed with Salkowski colorimetric solution. The mixture was then incubated in the dark for 30 min to allow for color development. King'B liquid medium and 50 mg / L IAA standard solution were used as negative and positive controls, respectively. The presence of IAA was initially determined by the color change (the mixture of IAA standard solution and colorimetric solution was pink, with a ratio of 1:1 > 2:1 > 3:1; the higher the IAA concentration, the darker the color, and the lower the concentration, the lighter the color). The experiment included 6 biological replicates.

[0046] (3) Detection results: After the fermentation broth of strain SY02 was mixed with the colorimetric solution and reacted in the dark for 30 min, the color of the mixture was significantly darker than that of the control, turning pink. Figure 4 f), proving that the strain has the ability to produce IAA.

[0047] Example 3 Preparation of Yano Sphingosine Monomer SY02 Fermentation Broth 1. Culture of strain SY02 on different solid culture media (1) Three solid culture medium formulations, including: PDA medium: Boil 200 g peeled potatoes in 1000 mL of water for 15 min, filter through four layers of gauze to remove potato residue, add 20 g glucose and 20 g agar powder, add water to make up to 1000 mL, and autoclave at 121℃ for 25 min.

[0048] R2A medium: 0.25 g / L tryptone + 0.5 g / L acid-hydrolyzed casein + 0.5 g / L soluble starch + 0.1 g / L magnesium sulfate + 12.0 g / L agar + 0.25 g / L peptone + 0.5 g / L yeast extract + 0.5 g / L glucose + 0.3 g / L dipotassium hydrogen phosphate + 0.3 g / L sodium pyruvate, autoclaved at 121℃ for 25 min.

[0049] Rice propagation medium: MS 4.4 g / L + 6-BA 1.2-1.5 mg / L + NAA 0.1-0.2 mg / L + agar 5.0 g / L + sucrose 30 g / L, pH 5.8-6.0, autoclaved at 121℃ for 25 min.

[0050] (2) Isolation, culture and identification of strain SY02 on different culture media Bacteria SY02 were picked from rice culture medium on a sterile operating table and inoculated onto PDA, R2A, and rice proliferation media for culture. Figure 5 After the bacteria emerged, their growth was observed. Results showed that SY02 grew better on PDA medium. Sanger bidirectional sequencing was performed on the colonies obtained on PDA medium using universal primers 16S-27F and 16S-1492R, and the obtained sequences were identical to the SY02 sequence. The results confirmed that the strain cultured on the PDA plate was *Sphingosine monocytogenes* SY02.

[0051] 2. Preparation of fermentation broth for Yano sphingosine strain SY02 (1) Three liquid culture medium formulations, including: PDA medium (1 L): 200 g peeled potatoes + 1000 mL water, boil for 15 min, filter through four layers of gauze to remove potato residue, add 20 g glucose, add water to make up to 1000 mL, autoclave at 121℃ for 25 min.

[0052] R2A medium (1 L): 0.25 g tryptone + 0.5 g acid-hydrolyzed casein + 0.5 g soluble starch + 0.1 g magnesium sulfate + 0.25 g peptone + 0.5 g yeast extract + 0.5 g glucose + 0.3 g dipotassium hydrogen phosphate + 0.3 g sodium pyruvate, autoclaved at 121°C for 25 min.

[0053] Rice propagation medium (1 L): MS 4.4 g + 6-BA 1.2-1.5 mg + NAA 0.1-0.2 mg + sucrose 30 g, pH 5.8-6.0, autoclaved at 121℃ for 25 min.

[0054] (2) Obtaining fermentation broth of strain SY02 in different liquid culture media Using an inoculation loop, scrape a sample of strain SY02 that has been cultured on a PDA plate for approximately 7-10 days and inoculate it into an Erlenmeyer flask containing 200 mL of liquid PDA. Place the flask in a shaker at 200 RPM and 28℃ for 24 h to obtain the seed culture of strain SY02. Add 1 mL of the seed culture to Erlenmeyer flasks containing 200 mL of PDA, R2A, and rice propagation medium, respectively. Measure the results at different time points using a microplate reader (0 h; 2 h; 4 h; 6 h; 8 h; 10 h; 12 h; 14 h; 16 h; 18 h; 20 h; 22 h; 24 h; 26 h; 28 h; 30 h; 32 h; 34 h; 36 h; 38 h; 40 h; 42 h; 44 h; 46 h; 48 h; 50 h; 52 h; 54 h; 56 h; 60 h; 64 h; 68 h; 72 h; 84 h; 96 h; 108 h). h; 120 h) Fermentation broth OD 600 The value was used to obtain the growth curve of strain SY02. Figure 6 The PDA culture time was determined to be 4–26 hours as the exponential growth period, and the OD... 600 The values ​​ranged from 0.09 to 1.33, with a stationary phase of 24 to 56 hours; the culture time for R2A was 4 to 14 hours, and the OD... 600 The OD value was 0.10–0.56, with a stationary period of 24–56 h; the culture time in the rice propagation medium was 4–20 h, and the OD value was... 600 The OD value was stable between 0.060 and 0.075 within a range of 12–120 h, ranging from 0.047 to 0.064. Therefore, the fermentation broth of this bacterium was prepared using liquid culture methods with PDA and R2A. The culture time in PDA medium was 4–26 h, and the culture time in R2A medium was 4–14 h.

[0055] Example 4: Effects of *Sphingosporium yannoides* SY02 on rice seed germination and rice growth 1. Differences in germination among different rice seeds Different rice varieties have different seed germination rates. Previous experiments showed that rice ZH11 seeds are extremely difficult to germinate; when cultured in water at 25℃, the germination rate on the third day was only 6.7%, significantly lower than that of the rice variety LTH, which had a germination rate of only 11.4% ( ). Figure 7 ).

[0056] 2. The germination-promoting effect of strain SY02 on rice seeds. To investigate the effect of strain SY02 on rice seed germination, OD 600 The SY02 fermentation broth with a value of 1.3 were respectively the original solution, diluted 10 times, and diluted 10 times. 3 times, 10 5times, 10 7 times, 10 9 times, 10 11 Rice seeds of variety ZH11 were treated with a double concentration gradient, with sterile water treatment serving as a control. Specifically, 20 rice seeds were placed in each 9 cm Petri dish lined with two layers of filter paper, and three dishes were treated for each concentration gradient. 5 mL of the corresponding concentration gradient bacterial suspension was added to each Petri dish during treatment, while 5 mL of distilled water was added to the blank control. The dishes were incubated at 25℃, and the number of germinating rice seeds was counted at different time points. The germination rate (%) was calculated as: number of germinating seeds / number of tested seeds. Root length and plant height were measured on day 24, and the number of roots was recorded. Data analysis was performed using one-way ANOVA (Analysis of Variance). p <0.05), data showed that on day 6 of rice seed germination, the germination rate of seeds treated with strain SY02 significantly increased by 2.0–11.5 times, and on day 8 by 1.4–2.9 times (Table 1); and on day 24 by 1.24–1.42 times (Table 1, Figure 8 Data show that strain SY02 not only significantly promoted the germination speed of rice seeds but also increased the final germination rate, with the optimal range being a 10:10 dilution. 5 Times—10 9 times.

[0057] The results of the survey on the number of roots, root length and plant height of rice seedlings showed that strain SY02 diluted 10 5 Times—10 9 The average number of roots was 1.45–1.87, which was 1.27 higher than the control; the average root length was 1.33–2.04 cm, which was 0.89 cm higher than the control; and the average plant height was 1.39–1.81 cm, which was 1.00 cm higher than the control, significantly promoting rice growth. Figure 8 ).

[0058] Table 1. Germination statistics of strain SY02 at different dilutions and treatment times for ZH11.

[0059] Example 5: Effect of *Sphingomonas yanoides* SY02 on promoting adventitious bud proliferation in tissue culture seedlings of different crops. 1. Effect of strain SY02 treatment on adventitious bud proliferation of aloe vera tissue culture seedlings Select aloe vera propagation seedlings with similar growth, cut them into individual plants, retaining a height of 2-3 cm, containing a growth point and two leaves, and inoculate them into aloe vera subculture medium (formulation: MS 4.4 g / L + agar 5.0 g / L + sugar 30.0 g / L + 6-BA 1.5-2.0 mg / L + NAA 0.05-0.1 mg / L, pH 5.8-6.0). After 5 days of culture, obtain contamination-free aloe vera subculture seedlings with uniform growth. Figure 6 A). 1 mL, 2 mL, and 3 mL of *Sphingosine monocytogenes* fermentation broth were added to tissue culture bottles containing aloe vera subculture seedlings of similar growth and without contamination. The seedlings were cultured for 60 days, and the adventitious bud proliferation rate of the aloe vera subculture seedlings was counted (n=25 plants per treatment). Subculture seedlings without fermentation broth served as a control (n=25 plants). Figure 9 Aloe vera (Table 2). The results showed that 1 mL, 2 mL, and 3 mL of fermentation broth from strain SY02 significantly promoted the proliferation of adventitious shoots of Aloe vera by 3.64±4.42 times, 4.00±3.73 times, and 1.88±1.66 times, respectively. The 1 mL and 2 mL treatments with strain SY02 showed significant differences compared to the control. p <0.05).

[0060] 2. Effect of treatment with strain SY02 on adventitious bud proliferation of strawberry tissue culture seedlings Select uniformly growing strawberry tissue culture clumps and cut them into individual plants 2-3 cm in height, containing a growing point and two leaves and a bud. Inoculate these plants into strawberry subculture medium (formulation: MS 4.4 g / L + agar 5.0 g / L + sugar 30.0 g / L + 6-BA 0.4-0.6 mg / L + NAA 0.1-0.15 mg / L, pH 5.8-6.0). Culture for 5 days to obtain uncontaminated, uniformly growing strawberry subcultures. Add 2 mL of *Sphingosine monocytogenes* fermentation broth to the tissue culture flasks containing similarly growing, uncontaminated strawberry subcultures and continue culturing for 60 days. Count the adventitious bud proliferation of the strawberry subcultures (n=125 plants), using subcultures without fermentation broth as a control (n=50 plants). Figure 9 Strawberry (Table 2). The results showed that strain SY02 promoted the proliferation of adventitious buds in strawberries by 7.68±4.67 times, which was significantly higher than the control (5.28±3.70 times). p <0.01).

[0061] 3. Effect of strain SY02 treatment on adventitious bud proliferation of *Amomum villosum* tissue culture seedlings Selected clumps of *Amomum villosum* tissue culture seedlings with similar growth vigor were cut into individual plants 2-3 cm in height containing growth points. These plants were then inoculated into *Amomum villosum* subculture medium (formulation: MS 4.4 g / L + agar 5.0 g / L + sugar 30.0 g / L + 6-BA 2.5-3.0 mg / L + NAA 0.1-0.2 mg / L, pH 5.8-6.0). After 5 days of culture, uncontaminated and uniformly growing *Amomum villosum* subculture seedlings were obtained. 2 mL of prepared *Sphingosine monocytogenes* fermentation broth was added to the tissue culture flasks containing these uniformly growing and uncontaminated subculture seedlings. Culture was continued for 60 days, and the adventitious bud proliferation rate of the subculture seedlings was counted (n=54 plants). Subculture seedlings without fermentation broth (n=27 plants) served as a control. Figure 9 (Table 2). The results showed that strain SY02 promoted the proliferation of adventitious shoots of cardamom by 2.50±1.54 times, which was significantly higher than the 1.41±0.62 times proliferation of the control shoots. p <0.001).

[0062] The OD600 values ​​of the fermentation broth of the SY02 strain of sphingosine monophosphate in Yano ranged from 0.1 to 1.33. This patent data statistically shows the OD600 values ​​of the SY02 fermentation broth. 600 The value was 1.23. The tissue culture conditions were as follows: culture temperature 28±2℃; light duration 12 h / d; light intensity 1600–2000 Lx. Shoot proliferation rate = number of shoots after treatment / number of inoculated shoots. Data are presented as mean ± standard deviation. The measured data were analyzed using SPSS 26.0 one-way ANOVA to test the significance level of differences between treatment groups.

[0063] Table 2. Effects of SY02 on bud proliferation in different crop species

[0064] Example 6: Mechanism of action of *Sphingosine monocytogenes* SY02 on promoting adventitious bud proliferation in *Aloe vera* tissue culture seedlings 1. Effects of strain SY02 on browning of aloe vera tissue culture seedlings Following the method described in Example 5, aloe vera tissue culture seedlings were cultured. On day 25 after treatment with fermentation broth from strain SY02, the browning degree of the aloe vera tissue culture seedlings was found to be lower than the control. Individual plant samples were collected. Figure 10 Therefore, the browning of the roots was graded and statistically analyzed based on the diameter of the browning. The results showed that the SY02 treatment significantly reduced the browning of the roots of Aloe vera tissue culture seedlings in the culture medium, with a reduction rate of 75.0%.

[0065] 2. Transcriptome sequencing and data analysis of aloe vera tissue culture seedlings Aloe vera plants were treated with 2 mL of fermentation broth from strain SY02, and single plants were used as controls. Aloe vera plant samples were collected on day 25 of treatment, with each treatment comprising 6 biological replicates. RNA extraction, library construction, and sequencing: Total RNA was extracted from plant tissues using the TRIzol method, and genomic DNA was removed using DNase I. RNA integrity was assessed using 1.0% agarose gel electrophoresis, RNA quality was determined using Bioanalyser 2100, and quantification was performed using ND-2000. High-quality RNA samples (OD) were selected. 260 / 280 =1.8~2.2, OD 260 / 230 Sequencing libraries were constructed using RNA with a molecular weight of ≥2.0 μg, RIN ≥8.0, 28S:18S ≥1.0, and >1 μg. RNA purification, reverse transcription, library construction, and sequencing were performed by Shanghai Meiji Biotechnology Co., Ltd., following the Illumina operating instructions. After the bidirectional RNA sequencing libraries passed the TBS380 test, they were sequenced using an Illumina NovaSeq 6000 sequencer.

[0066] Gene expression analysis: FASTP (https: / / github.com / OpenGene / fastp) was used to remove and quality control the raw data, obtaining Clean Reads. A reference-free genome assembly was performed, and Trinity was used to assemble all Clean Reads de novo. All genes and transcripts obtained from the transcriptome assembly were compared with six major databases (NR, Swiss-prot, Pfam, EggNOG, GO, and KEGG) to comprehensively obtain functional information of genes and transcripts and to statistically analyze the annotation status of each database. The TPM method was used to calculate the expression abundance of each gene.

[0067] Functional enrichment analysis: Differential expression analysis was performed using a combination of DESeq2 / DEGseq / EdgeR. Genes with |log2 FC| ≥ 1 and p-adjust ≤ 0.05 were defined as differentially expressed genes (DEGs). DEGs underwent GO functional gene enrichment analysis (Gene Ontology, http: / / www.geneontology.org) and KEGG pathway analysis (Kyoto Encyclopedia of Genes and Genomes, http: / / www.genome.jp / kegg / ). FDR < 0.05 indicated significantly enriched GO and KEGG genes. 3. Results of transcriptome data analysis Compared with the control, treatment of the fermentation broth of strain SY02 yielded 722 differentially expressed transcripts, including 577 transcripts with increased expression and 145 transcripts with decreased expression. GO functional enrichment analysis of these transcripts revealed that the differentially expressed transcripts were significantly enriched in GO components such as catalytic activity, polyketide biosynthetic process and polyketide metabolic process, naringenin-chalcone synthase activity, and secondary metabolite biosynthetic process and secondary metabolic process. Figure 11 KEGG analysis revealed that upregulated differentially expressed transcripts were significantly enriched in pathways including tropane, piperidine, and pyridine alkaloid biosynthesis; flavonoid biosynthesis; circadian rhythm; phenylpropanoid biosynthesis; the plant MAPK signaling pathway; pentose and glucuronate interconversions; plant-pathogen interaction; and alpha-linolenic acid metabolism. Figure 12 The study found that the flavonoid pathway included 21 highly expressed transcripts, whose expression levels were significantly higher than the control by 2.32–6.08 times (Table 3). The data indicate that treatment with the fermentation broth of strain SY02 primarily reduced browning and promoted the proliferation of adventitious shoots in aloe vera through flavonoid biosynthesis.

[0068] Table 3. Treatment of strain SY02 significantly increased gene expression levels in the flavonoid biosynthesis pathway.

[0069] Note: TPM stands for Transcripts Per Million, which is a measure of gene expression levels.

Claims

1. A rice endophytic *Sphingosphoensis sy02* strain, characterized by: The aforementioned Sphingosine monocytogenes ( Sphingobium yanoikuyae The accession number of SY02 is CCTCC NO: M 2026182, the accession date is January 22, 2026, and it is deposited at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China.

2. A biological agent, characterized in that, The biological agent comprises the fermentation broth of *Sphingosophytum yannoiense* SY02 and / or *Sphingosophytum yannoiense* SY02 as described in claim 1.

3. The biological agent according to claim 2, characterized in that, The biological agent has at least one of the following functions: (1) It produces indoleacetic acid, which promotes plant growth and seed germination; (2) Promote the proliferation of adventitious buds in tissue culture seedlings and alleviate browning of tissue culture seedlings.

4. The application of the rice endophytic sphingomyelin-yanosphingolipid SY02 according to claim 1 or the bacterial agent according to any one of claims 2 to 3 in promoting plant growth, seed germination, promoting the proliferation of adventitious buds in tissue culture seedlings, and preventing browning of tissue culture seedlings.

5. The biological agent according to claim 4, characterized in that, OD of the bacterial suspension of *Sphingosine monocytogenes* endophytic in rice 600 The range is 0.1 to 1.

33.

6. The application as described in claim 2, characterized in that: The method for preparing the fermentation broth of *Sphingosine monocytogenes* endophytic in rice includes the following steps: S1. Using an inoculation loop, scrape the bacterial strain cultured for 7-10 days from PDA and inoculate it into an Erlenmeyer flask containing 200 mL of liquid PDA. Place the flask on a shaker at 200 RPM and 28℃ for 24 h to obtain the seed culture of Yano sphingosine strain SY02. S2. Add 1 mL of the seed culture obtained in S1 to each of the conical flasks containing 200 mL of PDA and R2A medium, and culture for 4–26 h in PDA medium and 4–14 h in R2A medium.

7. The application as described in claim 6, characterized in that: The PDA culture medium is prepared by boiling 200 g of peeled potatoes in 1000 mL of water for 15 min, filtering the potato residue through four layers of gauze, adding 20 g of glucose, adding water to bring the volume to 1000 mL, and autoclaving at 121°C for 25 min. The R2A culture medium consisted of: 0.25 g / L tryptone, 0.5 g / L acid-hydrolyzed casein, 0.5 g / L soluble starch, 0.1 g / L magnesium sulfate, 0.25 g / L peptone, 0.5 g / L yeast extract, 0.5 g / L glucose, 0.3 g / L dipotassium hydrogen phosphate, and 0.3 g / L sodium pyruvate, and was autoclaved at 121°C for 25 min.

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