Transformed Ginsenoside Sphingosine Monoclonalella SP122 and its Application

Abstract

This invention relates to the field of microbial technology, and particularly to *Sphingomonas ginsenoside-transforming* SP122 and its applications. This invention isolates and obtains a strain of *Sphingomonas ginsenoside-transforming* SP122 that can effectively degrade the autotoxic substances of *Panax notoginseng*—saponins and phenolic acids. This strain can stably colonize the roots of *Panax notoginseng*, inhibit *Panax notoginseng* root rot pathogens, degrade crude saponins and generate new saponins, and degrade phenolic acids, thus alleviating the obstacle of continuous cropping of *Panax notoginseng*. Experimental verification shows that the application of this strain can improve the seedling survival rate and dry weight of *Panax notoginseng*, promote plant growth and dry matter accumulation, and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to ginsenoside-transforming Sphingosine Monoclonalella SP122 and its applications. Background Technology

[0002] Sanqi ( Panax. notoginseng Panax notoginseng (Burk.) FHChen is a perennial herb belonging to the genus Panax in the family Araliaceae. It is an important traditional Chinese medicine. However, due to the increasingly severe obstacles caused by continuous cropping of Panax notoginseng, the land resources suitable for its cultivation in traditional farmland have been significantly reduced. Root rot is the main manifestation of these obstacles, while autotoxicity may be the main contributing factor. Therefore, reducing the accumulation of autotoxic substances such as saponins in the soil after continuous cropping of Panax notoginseng is one of the effective ways to alleviate these obstacles.

[0003] Currently, the main strategies for addressing this problem include biochar adsorption, crop rotation, and microbial degradation. However, these measures to reduce autotoxic substances also have limitations. Specifically, the adsorption of autotoxic substances by biochar is mainly based on a reversible physical adsorption mechanism, which may lead to the re-release of adsorbed substances back into the soil environment under certain conditions, thus affecting its long-term effectiveness. On the other hand, while crop rotation can effectively mitigate autotoxicity, its practical application is often limited by a long implementation period, which may be detrimental to the efficient operation of agricultural production. Furthermore, although introducing exogenous microbial strains capable of degrading saponin-type autotoxic substances into continuously cropped soils is considered a highly promising biological strategy, it still faces many challenges in practical application. Specifically, the colonization efficiency of these exogenous strains in the soil may be low, resulting in insufficient surviving numbers and thus affecting their degradation efficiency. Moreover, even if successful colonization occurs, their degradation efficiency may be affected by various factors such as soil environment and competition from indigenous microorganisms, thus limiting their widespread application in actual production. Therefore, given the existing challenges in pathogen and autotoxic substance reduction strategies, exploring efficient and non-chemically dependent biological control methods has become an urgent need.

[0004] The autotoxic substances in Panax notoginseng mainly include saponins and phenolic acids. Therefore, how to provide a microorganism that can effectively degrade saponins and phenolic acids in Panax notoginseng for biological control has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] First, this invention provides a strain of *Sphingosine Monoclonalella* that transforms ginsenosides (… Sphingomonas ginsenosidimutans The strain is *Sphingosine Monoclonalis* SP122, which is a transgenic strain of ginsenoside Sphingosine, and its accession number is CGMCC No. 30965.

[0006] The aforementioned *Sphingosine Transformer* SP122 was screened from the rhizosphere soil of *Panax notoginseng*. This strain was deposited on June 14, 2024, at the China General Microbiological Culture Collection Center (CGC-MSC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. Its classification and naming are: *Sphingosine Transformer*. Sphingomonas ginsenosidimutans The accession number is CGMCCNo.30965.

[0007] Secondly, the present invention provides a microbial inoculant comprising the aforementioned transformed ginsenoside sphingosine monocytogenes SP122.

[0008] In some embodiments, the microbial agent is a freeze-dried fermentation broth of *Sphingosine Monomer* SP122 that has been transformed with ginsenosides. The freeze-dried fermentation broth contains *Sphingosine Monomer* SP122.

[0009] In some embodiments, the freeze-dried fermentation broth powder also includes a freeze-drying protectant.

[0010] Specifically, the freeze-drying protectant includes, but is not limited to, skim milk powder, dextrin, lactose, and sucrose.

[0011] Thirdly, the present invention provides the application of the transformed ginsenoside sphingosine monocytogenes SP122 or the microbial agent in inhibiting root rot pathogens.

[0012] Preferably, the root rot pathogen is pathogen LD ( Fusarium solani ).

[0013] Preferably, the inhibitory effect on root rot pathogens is enhanced by using Panax notoginseng crude saponins as a carbon source.

[0014] Fourthly, the present invention provides the application of the aforementioned ginsenoside-transforming Sphingosine Monoclonalella SP122 or the aforementioned microbial agent in regulating the degradation or generation of Panax notoginseng crude saponins.

[0015] Fifthly, the present invention provides the application of the aforementioned transformed ginsenoside sphingosine monocytogenes SP122 or the aforementioned microbial agent in improving the survival rate of Panax notoginseng seedlings or the dry weight of Panax notoginseng.

[0016] In a sixth aspect, the present invention provides the application of the aforementioned transformed ginsenoside sphingosine monocytogenes SP122 or the aforementioned microbial agent in the degradation of phenolic acids.

[0017] Preferably, the phenolic acid is p-hydroxybenzoic acid, ferulic acid, coumaric acid, or cinnamic acid.

[0018] In a seventh aspect, the present invention provides the application of the transformed ginsenoside sphingosine monocytogenes SP122 or the microbial agent in promoting plant growth or dry matter accumulation; preferably, the plant is a plant with continuous cropping obstacles; preferably, the plant is lettuce, tomato, strawberry or soybean.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention isolates and obtains a strain of *Sphingosine Monoclonalella* SP122 that can effectively degrade the autotoxic substances of Panax notoginseng—saponins and phenolic acids. This strain can stably colonize the roots of Panax notoginseng, inhibit root rot pathogens, degrade crude saponins and generate new saponins, and degrade phenolic acids, thus alleviating the obstacle of continuous cropping of Panax notoginseng. Experimental verification shows that the application of this strain can improve the survival rate and dry weight of Panax notoginseng, promote plant growth and dry matter accumulation, and has broad application prospects. Attached Figure Description

[0021] Figure 1 This is a culture photograph of *Sphingosine Monoclonalis* SP122 transformed with ginsenosides.

[0022] Figure 2 It is a phylogenetic tree.

[0023] Figure 3 This is a graph showing the results of the identification of the ability of *Sphingosine Monoclonalis* SP122 to produce β-glucosidase.

[0024] Figure 4 This is a diagram showing the results of an experiment on the inhibition of root rot pathogens by *Sphingosine Monoclonalella* SP122 transformed with ginsenosides.

[0025] Figure 5 This is a growth curve of *Sphingosine Monoclonalis* SP122 transformed with ginsenosides.

[0026] Figure 6 This is a graph showing the survival rate of seedlings in an indoor potted plant experiment.

[0027] Figure 7 This is a graph showing the results of the single-plant dry weight of the indoor potted plant experiment.

[0028] Figure 8 This is a graph showing the degradation results of crude saponins in the culture medium by *Sphingosine Monoclonalella* SP122, which transforms ginsenosides.

[0029] Figure 9 This is a diagram showing the degradation results of saponins in continuously cropped soil by *Sphingosine Monoclonalella* SP122, which transforms ginsenosides.

[0030] Figure 10 This is a graph showing the germination results of Panax notoginseng seedlings under forest cover after treatment with Sphingomyelin-transformed ginsenoside SP122.

[0031] Figure 11 This is a graph showing the degradation results of phenolic acids by *Sphingosine Monoclonalis* SP122, which transforms ginsenosides.

[0032] Figure 12 This is a graph showing the effect of transforming ginsenoside sphingosine monocytogenes SP122 on lettuce growth.

[0033] Figure 13 This is a graph showing the effect of transforming ginsenoside sphingosine monocytogenes SP122 on tomato growth.

[0034] Figure 14 This is a graph showing the effect of transforming ginsenoside sphingosine monocytogenes SP122 on strawberry growth.

[0035] Figure 15 This is a graph showing the effect of transforming ginsenoside sphingosine monocytogenes SP122 on soybean growth. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. In statistical analysis, different asterisks represent significant differences, * p <0.05,** p <0.01, *** p <0.001.

[0037] The culture medium formulation using crude saponins as the sole carbon source in the following examples is as follows: 3g NaNO3, 1g KH2PO4, 0.5g KCl, 0.5g MgSO4•7H2O, 0.01g FeSO4•7H2O, 10g Panax notoginseng crude saponins, 17g agar, 1000mL distilled water. Dissolve and mix well, then adjust the pH to 7.0 with 1mol / L NaOH solution. The crude Panax notoginseng saponins were purchased from Shaanxi Jinkangtai Biotechnology Co., Ltd.

[0038] Example 1: Isolation of Ginsenoside Sphingosine Monoclonal Antibacterial Peptidobacterium SP122

[0039] Soil microorganisms were isolated from the rhizosphere soil of *Panax notoginseng* stored at 4°C using a culture-isolation method. Fungi were isolated using Bengal Red and PDA media, while bacteria were isolated using LB, NA, R2A, and modified Gao's No. 1 media (Wang et al., 2020; van der Linde et al., 1999; Lin et al., 2012). Following the principles of cultinomy, four media—NA, LB, R2A, and PDA—were selected for the isolation and culture of *Panax notoginseng* rhizosphere soil microorganisms. 1 g of each soil sample stored at 4°C was placed in a 15 mL sterile centrifuge tube, and 9 mL of sterile distilled water was added. The mixture was shaken at 120 rpm for 30 min and then allowed to stand for 2 min to prepare a soil microbial suspension. The soil microbial suspension was diluted to 10⁻¹⁰ with sterile water. 3 10 4 and 10 5 100 μL of each culture medium was added to the surface of different solid culture media, spread evenly with a spreader, and incubated in a 28°C incubator. Single colonies were picked from the corresponding plates at different incubation times (3-7 days) and purified on NA (bacteria) / PDA (fungi) medium. After three purifications, when no contaminating bacteria appeared, single colonies were picked, preserved, and sequenced. Fungi were amplified and sequenced using ITS (Guo Penghao et al., 2012), and bacteria were amplified and sequenced using 16S rDNA (Cai et al., 2012). The sequencing results were compared using BLAST in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ).

[0040] A microorganism was isolated, and the streak isolation photo is shown below. Figure 1 As shown, the 16S sequence of this bacterium was determined, and after alignment on the NCBI platform, a phylogenetic tree was constructed, as follows. Figure 2 As shown, the bacterium was identified as *Sphingosine Transformans*. Sphingomonas ginsenosidimutans It was named Transgenic Ginsenoside Sphingosine Monoclonalis SP122, with accession number CGMCC No.30965.

[0041] Furthermore, the ability to produce β-glucosidase was identified. Aescin is a white needle-like crystal that is hydrolyzed by β-glucosidase produced by microbial metabolism, yielding aescin and glucose. Aescin reacts with iron ions in the culture medium to form a black substance. The experimental results are shown below. Figure 3 The aescin medium turned black, indicating that the transformed ginsenoside sphingosine monocytogenes SP122 can produce β-glucosidase. CK is the sterile water control group.

[0042] Example 2: Inhibition of pathogens by *Sphingosine Monoclonalella* SP122 transformed with ginsenosides on crude saponin plates.

[0043] This embodiment observed the inhibitory effect of *Sphingosine Monoclonalella* SP122, transformed with different concentrations of crude saponins, on root rot pathogens on plates. The test results are as follows: Figure 4 As shown, the pathogenic bacteria LD ( Fusarium solani The colony radius was significantly inhibited in plates with different concentrations of crude saponins, and the inhibitory effect increased with increasing crude saponin concentration. Co-culture experiments of *Sphingomyelin-transformed* SP122 and *LD* showed that SP122 had a strong antagonistic effect on LD, and the colony diameter decreased significantly with increasing crude saponin concentration, indicating that SP122's inhibitory effect on LD increased.

[0044] To investigate the degradation effect of ginsenoside-transforming Sphingomonas SP122 on crude saponins, an experiment was also conducted using crude saponins as the sole carbon source. The results are as follows: Figure 4 As shown, crude saponins significantly promoted the growth of ginsenoside-transforming Sphingosine Monoclonal strain SP122, with CK being the sterile water control group.

[0045] Example 3: Effects of crude saponins on the growth and biofilm formation of Ginsenoside-converting Sphingosine Monoclonalella SP122

[0046] In this embodiment, *Sphingosine Monoclonalis* SP122 transformed with crude saponins as the sole carbon source was cultured. The transformed *Sphingosine Monoclonalis* SP122 was cultured in LB medium, and the OD590 value was measured using a microplate reader. The culture was diluted to approximately 0.4, and 100 μL of the bacterial culture was added to 200 mL of liquid culture medium with crude Panax notoginseng saponins as the sole carbon source (10 mg / L). A control group without added bacterial culture was used. The plates were incubated on a shaker (28℃, 150 r / min). Every 3 hours, 200 μL was transferred to 96-well plates, and the OD590 was measured at 590 nm using a microplate reader. Bacterial growth curves were plotted. Simultaneously, the effect of crude saponins on bacterial biofilm was determined using the crystal violet method (Wang Lijun et al., 2019).

[0047] In a culture medium with crude saponins as the sole carbon source, the bacterial growth curve is as follows: Figure 5 As shown, with prolonged culture time, the bacterial strain grew, and the OD value of the bacterial solution increased, indicating that crude saponins promoted the growth of *Sphingosine Monoclonalella* SP122. This suggests that *Sphingosine Monoclonalella* SP122 can tolerate crude saponins, and that crude saponins from Panax notoginseng can promote the vegetative growth of the strain, allowing it to occupy a larger ecological niche in the roots of Panax notoginseng.

[0048] Furthermore, *Sphingosine Monoclonalis* SP122 transformed with ginsenosides was inoculated into a 96-well plate containing a medium with crude saponins as the sole carbon source. After incubation at 28°C for 52 hours, the biofilm content was determined using the crystal violet method, and the OD490 value was read. Figure 5As shown, the biofilm amount of *Sphingomonas ginsenoside SP122* transformed with ginsenosides was significantly higher than that of CK (0 g / L, sterile water), indicating that crude saponins promoted biofilm formation of *Sphingomonas ginsenoside SP122* and enhanced its colonization in the roots of *Panax notoginseng*.

[0049] Example 4: Effect of Transformation of Ginsenoside Sphingosine Monoclonalis SP122 on the Growth of Panax notoginseng

[0050] A certain amount of crude Panax notoginseng saponins was added to a small basin containing 1 kg of forest soil and mixed thoroughly to achieve a saponin content of 10 ppm in the soil, simulating the total saponin concentration in three-year-old Panax notoginseng soil (Wang Luotao, 2020). The mixture was left to stand for 3 days to obtain exogenously added crude Panax notoginseng saponin soil. Soil from continuous cropping (where Panax notoginseng had been grown for three years in a greenhouse in Xundian) was mixed with treated unplanted forest soil at a 1:1 ratio and left to stand for 3 days to obtain continuous cropping soil.

[0051] Select healthy Panax notoginseng seeds, surface disinfect them with 1.5% NaClO for 5 minutes, rinse them three times with sterile water, and blot dry with sterile filter paper. Use 10 seeds per pot, with 10 replicates per treatment. In the laboratory, NA liquid culture medium for transforming ginsenoside sphingosine monocytogenes SP122 was prepared and shaken to achieve an OD value of approximately 0.4. After complete emergence, each pot was irrigated with 50 mL of culture medium (culture medium = 1 part 5 mL bacterial suspension : 9 parts sterile water). Sterile water was used as the control group (CK). The treatment groups included four groups, as follows: Soil from forests where no Panax notoginseng was planted was collected and divided into two portions. One portion was sterilized by steam at 100℃ for 4 hours and then allowed to cool down for later use (referred to as sterilized). The other portion was not sterilized (referred to as unsterilized). The two soil portions were further treated. One portion was treated by adding crude saponins at a concentration of 10 mg / kg, mixing thoroughly, and then potting it (15 cm diameter flowerpot, approximately 1 kg of soil per pot) (referred to as saponin). The other portion was treated by adding 50% Panax notoginseng continuous cropping soil (soil from Panax notoginseng continuously planted for three years) + 50% of the above-treated forest soil (sterilized / unsterilized), mixing thoroughly, and then potting it (15 cm diameter flowerpot, approximately 1 kg of soil per pot) (referred to as 50% continuous cropping soil). Planting Panax notoginseng seeds. After potted planting, the seeds are randomly placed in the greenhouse at the Xundian base and managed routinely. Irrigate once a week for two consecutive months, and then count the seedling survival rate. After the Panax notoginseng continues to grow for another two months, collect samples of the potted Panax notoginseng, clean them, sterilize them at 115℃ for 30 minutes, dry them at 60℃ for 3 days, weigh them, and calculate the dry weight of each plant.

[0052] The seedling survival rate results of the indoor pot experiment are as follows: Figure 6As shown, *Sphingomonas ginsenoside SP122* significantly improved the seedling survival rate of *Panax notoginseng* in unsterilized soil with added saponins, but there was no significant difference after sterilization. *Sphingomonas ginsenoside SP122* significantly improved the seedling survival rate of *Panax notoginseng* in continuously cropped soil. In summary, *Sphingomonas ginsenoside SP122* can significantly improve the seedling survival rate of *Panax notoginseng*, and the effect is better in continuously cropped soil and unsterilized soil. This indicates that *Sphingomonas ginsenoside SP122* can promote the growth of *Panax notoginseng* in soil containing initial microorganisms, especially when saponins are present in the soil. This suggests that *Sphingomonas ginsenoside SP122* can not only degrade saponins but may also function more as a recruiter of beneficial microorganisms.

[0053] The results of the single-plant dry weight of the indoor potted plant experiment are as follows: Figure 7 As shown, transformation of Ginsenosphingomonas SP122 significantly increased the dry weight of individual plants in all four treatments. Transformation of Ginsenosphingomonas SP122 can significantly promote the accumulation of Panax notoginseng dry matter, especially when the continuous cropping is aggravated.

[0054] Example 5: Study on the degradation of crude saponins in culture medium by *Sphingosine Monoclonalella* SP122 transformed with ginsenosides.

[0055] 500 μL of NA culture broth from *Sphingomonas sp.* SP122, transformed with ginsenosides, was added to 50 mL of a single carbon source culture broth containing 1 g / L crude saponins. After incubation at room temperature for 72 h, 2 mL of the liquid was collected and freeze-dried for 48 h (-40℃, 0.12 kPa). An equal volume of 75% chromatographic methanol was added to the freeze-dried material to dissolve it, and the mixture was thoroughly mixed by pipetting. 1 mL of the methanol solution was placed in a 1.5 mL centrifuge tube and centrifuged at 14000 rpm for 15 min. The supernatant was collected and analyzed by HPLC. The contents of 12 saponins (R1, Rg1, Re, Rb1, Rd, Rf, Rb2, Rh1, Rc, Rh2, Rb3, and Rg3) in the crude saponins were determined by HPLC. The HPLC detection steps are as follows:

[0056] (1) HPLC detection conditions were as follows: column (4.6 mm × 150 mm, 4 µm, Agilent Poroshell 120EC-C18); mobile phase A: acetonitrile; mobile phase B: water; flow rate 1.0 mL / min; column temperature 30℃; detection wavelength 203 nm; injection volume 10 µL. Elution gradient: 0-20 min, 82% B; 20-40 min, 82-57% B; 40-48 min, 57-45% B; 48-54 min, 45% B; 54-56 min, 45-5% B; 56-71 min, 5% B; 71-72 min, 5-82% B; 72-74 min, 82% B. Under these conditions, baseline separation of 12 major saponins could be achieved.

[0057] (2) Prepare standard solutions of 12 saponins. Accurately weigh the corresponding standard into a 2 mL volumetric flask and dilute to volume. Dilute the solution with methanol in a certain proportion to prepare standard solutions of 5 ppm, 10 ppm, 25 ppm, 50 ppm, 100 ppm, 200 ppm and 500 ppm. Filter the obtained standard solutions through a 0.22 μm filter membrane for later use.

[0058] (3) Determination of linearity. HPLC was used for detection, and a standard curve was plotted with the peak area (Y) as the ordinate and the sample concentration (X) as the abscissa.

[0059] (4) Weigh 0.2 g of crude saponin, dissolve it in 75% chromatographic methanol, sonicate for 30 min, centrifuge at 10000 rpm for 10 min, filter the solution through a 0.22 μm filter membrane, detect it by HPLC, and determine the concentration of each monomer saponin in the crude saponin according to the standard curve. Calculate the saponin conversion rate based on the concentration of each monomer saponin in the uninoculated bacteria.

[0060] Test results are as follows Figure 8As shown, the saponin content in the solution changed after inoculation with *Sphingomonas ginsenoside* SP122, indicating that *Sphingomonas ginsenoside* SP122 significantly degraded crude saponins of Panax notoginseng. The total saponin content was significantly lower than that of the blank treatment. Detailed results are as follows: Among the *Sphingomonas ginsenoside* SP122, the average degradation rates of Rg1 (86.1%), Rh1 (57.77%), and Rf (36.33%) were above 10%, while the degradation rates of other saponins were lower (Rb1: 9.60%, Rb2: 5.77%, R1: 3.81%, Re: 3.23%, Rd: 7.11%, Rc: 2.53%). Rg2 was also generated (generation rate 100.8%). In addition, three saponins were generated: Rh2 (at a concentration of 0.04 mg / g), Rg3 (at a concentration of 0.17 mg / g), and Rb3 (at a concentration of 1.44 mg / g).

[0061] Comprehensive analysis results show that the ginsenoside-transforming Sphingosine Monoclonalella SP122 can not only degrade saponins, but also generate new saponins.

[0062] Example 6: Study on the effects of ginsenoside-transforming Sphingosine Monoclonalella SP122 on saponin degradation in continuously cropped soil and on the emergence rate of Panax notoginseng seedlings under forest cover.

[0063] To investigate the role of *Sphingomonas SP122*, which transforms ginsenosides, in soil, a study on soil saponin degradation was conducted. Soil under trees that had been continuously cropped with *Panax notoginseng* for three years was selected for bacterial treatment. The detailed treatment process was as follows: A NA liquid culture medium for *Sphingomonas SP122* was prepared in the laboratory and cultured by shaking until the bacterial OD value was approximately 0.4. Then, the culture was prepared at a concentration of 200 mL / m³. 3 The bacterial count was used to irrigate the soil under the continuous forest. After irrigation, the treated soil was mixed evenly. The treatment was carried out once a week for eight consecutive weeks, for a total of two months. Before each treatment, soil samples were taken for quantitative analysis of soil saponins to calculate the saponin degradation rate.

[0064] The results are as follows Figure 9 As shown (CK represents the sterile water treatment group, SP represents the group treated with *Sphingomonas ginsenoside* SP122), *Sphingomonas ginsenoside* SP122 degraded five major saponins in *Panax notoginseng* continuously cropped soil to varying degrees. Among these, SP showed higher initial degradation of R1 and Rg1 than the control, with a more significant difference in degradation effect later. For Rb1 and Rg1... d and R eAmong the five saponins, SP showed a significantly higher degradation effect than CK. The combined results of the five saponin studies indicated that SP's degradation capacity was significantly higher than CK. The soil saponin degradation effect clearly demonstrated the ability of *Sphingomonas ginsenoside-transforming* SP122 to degrade saponins, providing important evidence for the study of the degradation capacity of *Sphingomonas ginsenoside-transforming* SP122.

[0065] Furthermore, after two months of soil treatment, *Panax notoginseng* twigs and seeds were planted under the forest canopy to study the germination rate of *Panax notoginseng* seedlings under the forest canopy after treatment with *Sphingomonas ginsenoside-transformed* SP122. The results showed that ( Figure 10 Treatment with *Sphingomyelin-transformed* SP122 significantly improved the germination rate of one-year-old and two-year-old *Panax notoginseng* plants under forest cover. Therefore, in forest environments, treatment with *Sphingomyelin-transformed* SP122 can significantly improve the germination rate of both one-year-old and two-year-old *Panax notoginseng*, with a more significant effect on two-year-old plants, indicating that *Sphingomyelin-transformed* SP122 has greater application potential in actual production.

[0066] Example 7: The effect of transforming ginsenoside sphingosine monocytogenes SP122 on the degradation of phenolic acids

[0067] Numerous studies have reported that *Sphingomonas* can degrade various polycyclic aromatic hydrocarbons and phenolic substances, and can survive in nutrient-poor and harsh conditions, exhibiting good environmental adaptability and tolerance. It has been reported that *Sphingomonas* can be used to produce carotene and bioglue. Phenolic acids secreted by plant roots are the main substances causing root autotoxicity, with the soluble portion and reversibly adsorbed components of phenolic acids being directly related to autotoxicity. Phenolic acids, such as benzoic acid, salicylic acid, and p-hydroxybenzoic acid, have been detected in the root exudates of continuously planted peanuts, melons, tobacco, and cucumbers. Phenolic acids have a significant inhibitory effect on seed germination and plant growth. Previous research by our group found that the accumulation of phenolic acids is also an important cause of continuous cropping obstacles in *Panax notoginseng*. Therefore, this example tests the degradation effect of *Sphingomonas* SP122, which transforms ginsenosides, on phenolic acids.

[0068] Soil was treated with *Sphingomonas ginsenoside-transforming* SP122 for two consecutive months, with weekly irrigation. Each time, the OD value of the bacterial solution was 0.6, and 50 ml was used. Sterile water treatment served as the control group (CK). The initial and final contents of four phenolic acids in the soil were determined by absolute quantification. The degradation effects of bacteria on the four main phenolic acids in the soil are summarized below. Figure 11In p-hydroxybenzoic acid, the average degradation rate of the CK group was 5.03%. The degradation ability of the group treated with *Sphingomyelin-transformed* SP122 was stronger than that of the control, with an average degradation rate of 26.33%. In ferulic acid, the average degradation rate of the group treated with *Sphingomyelin-transformed* SP122 was 15.71%. In p-coumaric acid, the average degradation rate of the CK group was 2.40%, while the degradation ability of *Sphingomyelin-transformed* SP122 was the strongest, with an average degradation rate of 6.96%. In cinnamic acid, which had the lowest content in the soil, the average degradation rate of *Sphingomyelin-transformed* SP122 was 0.62%. Therefore, the average degradation rate of the four phenolic acids in the soil by the CK group was 5.02%, while the degradation rate by *Sphingomyelin-transformed* SP122 was 17.27%. Comprehensive analysis showed that *Sphingosine Monoclonalis* SP122, which transforms ginsenosides, can significantly degrade phenolic acids in the soil, but the degradation capacity for different phenolic acids varies.

[0069] Example 8: Effects of Transformation of Ginsenoside Sphingosine Monoclonalis SP122 on Plant Growth

[0070] 1. Effects of Ginsenoside-transformed Sphingosine Monoclonalella SP122 on lettuce growth

[0071] To explore the effects of ginsenoside-transformed Sphingomonas SP122 on plant growth, lettuce, which is sensitive to phenolic acids, was selected for growth measurement. Lettuce seeds were planted in soil that had been treated with ginsenoside-transformed Sphingomonas SP122 (treatment method as in Example 7) for two months. The survival rate, fresh weight per plant, dry weight per plant, and root growth of lettuce were recorded.

[0072] The results show that ( Figure 12 Treatment with *Sphingomyelin-mediated ginsenoside SP122* did not significantly affect the survival rate of lettuce seedlings; however, it significantly increased the fresh weight and dry weight per lettuce plant. Furthermore, *Sphingomyelin-mediated ginsenoside SP122 treatment significantly increased root length, root surface area, and root volume. Therefore, treatment with *Sphingomyelin-mediated ginsenoside SP122* promoted lettuce growth and dry matter accumulation.

[0073] 2. Effects of Ginsenoside-Transforming Sphingosine Monoclonal Antibacterial Bacterium SP122 on Tomato Growth

[0074] Studies have shown that phenolic acids affect the growth of tomato seedlings. Therefore, tomatoes were selected for growth measurement. Tomato seeds were planted in soil that had been treated with ginsenoside sphingomyelin-transformed Sphingosine Monoclonalis SP122 (treatment method as in Example 7) for two months. The survival rate of tomato seedlings, fresh weight per plant, dry weight per plant and root growth were recorded.

[0075] The results show that ( Figure 13 Treatment with *Sphingomyelin-mediated transformation* SP122 significantly increased the seedling survival rate of tomatoes; it also significantly increased the fresh weight and dry weight per plant; and it significantly increased the root length, root surface area, and root volume. Therefore, treatment with *Sphingomyelin-mediated transformation* SP122 promoted the growth and dry matter accumulation of tomatoes.

[0076] 3. Effects of Ginsenoside-Transforming Sphingosine Monoclonal Antibacterial Bacterium SP122 on Strawberry Growth

[0077] Studies have shown that the accumulation of phenolic acids in the soil is an important cause of continuous cropping obstacles in strawberries. In order to study the degradation of phenolic acids by Sphingomyelin SP122, strawberries were selected for pot verification. Strawberry seedlings were transplanted into the soil treated (treatment method is the same as in Example 7) for two months. After the growth differences were observed, the survival rate, fresh weight of single plant, dry weight of single plant and growth of new roots were counted.

[0078] The results show that ( Figure 14 Treatment with *Sphingomyelin-transformed* SP122 significantly increased the survival rate of strawberry seedlings; it also significantly increased the fresh weight and dry weight of individual strawberry plants. Among the four root system indicators, the *Sphingomyelin-transformed* SP122 treatment was slightly higher than the control, but the difference was not statistically significant. Therefore, treatment with *Sphingomyelin-transformed* SP122 promoted the growth of strawberry aboveground parts.

[0079] 4. Effects of Ginsenoside-Transforming Sphingosine Monoclonal Antibacterial Bacterium SP122 on Soybean Growth

[0080] Studies have shown that the accumulation of phenolic acids in the soil affects the growth of soybean seedlings. Therefore, soybeans were selected for pot experiments. Soybean seeds were planted in soil that had been treated (treatment method is the same as in Example 7) for two months. After the differences in growth were observed, the survival rate, fresh weight of single plants, dry weight of single plants and growth of new roots were statistically analyzed.

[0081] The results show that ( Figure 15 There was no significant difference in soybean seedling survival rate between the control (CK) and the control (control) after treatment with *Sphingomonas ginsenoside* SP122. However, among the four root indicators, there was a significant difference between the control and CK after treatment with *Sphingomonas ginsenoside* SP122, indicating that the *Sphingomonas ginsenoside* SP122 treatment increased soybean root length and root volume. Therefore, treatment with *Sphingomonas ginsenoside* SP122 promoted soybean growth.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of *Sphingosine Monoclonalella* that transforms ginsenosides ( Sphingomonas ginsenosidimutans ), characterized in that, It is transformed ginsenoside sphingosine monocytogenes SP122, with accession number CGMCC No.30965.

2. A microbial agent containing the ginsenoside-transforming sphingosine monocytogenes as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The microbial agent is a freeze-dried fermentation broth of *Sphingosine Monoclonalella* SP122 that transforms ginsenosides.

4. The microbial agent according to claim 3, characterized in that, The freeze-dried fermentation broth also includes a freeze-drying protectant.

5. The application of the *Sphingomonas ginsenoside-transforming* strain according to claim 1 or the microbial agent according to any one of claims 2-4 in inhibiting root rot pathogens, wherein the root rot pathogen is... Fusarium solani .

6. The application according to claim 5, characterized in that, The inhibitory effect on the root rot pathogen was enhanced by using Panax notoginseng crude saponins as a carbon source.

7. The application of the *Sphingomonas* strain transforming ginsenosides according to claim 1 or the microbial agent according to any one of claims 2 to 4 in improving the survival rate of Panax notoginseng seedlings or the dry weight of Panax notoginseng.

8. The use of the ginsenoside-transforming sphingosine monocytogenes according to claim 1 or the microbial agent according to any one of claims 2 to 4 in the degradation of Rg1, Rh1, Rf, Rb1, Rb2, R1, Re, Rd or Rc saponins or in the generation of Rg2, Rh2, Rg3 or Rb3 saponins.

9. The application of the *Sphingomonas* strain transforming ginsenosides according to claim 1 or the microbial agent according to any one of claims 2-4 in the degradation of phenolic acids, characterized in that... The phenolic acid is p-hydroxybenzoic acid, ferulic acid, coumaric acid, or cinnamic acid.

10. The application of the *Sphingomonas ginsenoside-transforming* strain according to claim 1 or the microbial agent according to any one of claims 2-4 in promoting plant growth or dry matter accumulation, characterized in that, The plant is lettuce, tomato, strawberry or soybean.

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