Application of rahnella maple XS-11 in root system growth promotion of radix bupleuri

Abstract

The invention provides application of rahnella maple XS-11 in the aspect of growth promotion of radix bupleuri, and belongs to the technical field of microorganisms. The invention provides rahnlaaceae XS-11, and the collection number of the rahnlaaceae XS-11 is CGMCC (China General Microbiological Culture Collection Center) No.35248. The rahnlaaceae XS-11 can be used for preparing a culture medium. In the invention, the bacterium rahnella malayi XS-11 is a growth-promoting bacterium screened from rhizosphere soil in a bupleurum chinense genuine producing area, the XS-11 strain has the capabilities of dissolving phosphorus, dissolving potassium and fixing nitrogen, can promote the growth of bupleurum chinense seedlings as a growth promoting agent, particularly has a remarkable effect of increasing the biomass of the root of a medicine part, and meanwhile, has a remarkable effect of promoting the growth of the bupleurum chinense seedlings. The growth-promoting bacterium can also significantly improve accumulation of saponin components at the root of radix bupleuri, can be used for artificial cultivation of radix bupleuri, provides a theoretical basis for increasing the yield and improving the quality of radix bupleuri, and has important guiding significance for reasonably utilizing biological agents to carry out ecological planting of medicinal plants.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine cultivation and genetic engineering technology, and particularly relates to application of Rahnellaaceris XS-11 in promoting growth of radix bupleuri root system. BACKGROUND

[0002] Radix bupleuri (Bupleurum chinense L.) is a plant of the genus Bupleurum in the family Umbelliferae, and its dried root is a commonly used traditional Chinese medicine. Radix bupleuri is mainly produced in Henan, Shaanxi, Shanxi and other places, and is mostly found in mountain slope grasslands, forest edges or shrubs. Radix bupleuri has bitter taste and slightly cold nature, and is related to the liver and gallbladder channels, and has the functions of relieving the exterior and reducing fever, soothing the liver and relieving depression, and lifting yang qi. Modern research has found that radix bupleuri mainly contains saikosaponins, volatile oils and other components. Among them, saikosaponins are the main effective components, and have anti-inflammatory, antiviral, liver-protecting, antipyretic and other pharmacological effects.

[0003] In radix bupleuri cultivation, due to regional conditions, pests and diseases and other reasons, the plant growth is often slow and the active ingredient content is low. The rhizosphere bacteria XS-11 screened in the present application can promote the growth of radix bupleuri to some extent and significantly increase the content of active ingredients (saikosaponins a, b2, d and f). SUMMARY

[0004] The present application aims to solve the problems in the prior art, and provides application of Rahnellaaceris XS-11 in promoting growth of radix bupleuri root system.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical scheme:

[0006] The application of Rahnellaaceris XS-11 in promoting growth of radix bupleuri, wherein the classification name of the Rahnellaaceris XS-11 is Rahnellaaceris, which is preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Haidian District, Beijing, with the preservation number of CGMCC No. 35248 and the preservation date of July 16, 2025;

[0007] The application refers to applying the Rahnellaaceris XS-11 or a bacterial agent containing the Rahnellaaceris XS-11 to radix bupleuri seedlings.

[0008] Preferably, the bacterial agent refers to a bacterial suspension containing the Rahnellaaceris XS-11 with a dosage of OD600 = 0.4.

[0009] The application includes application of the Rahnellaaceris XS-11 or the bacterial agent in promoting growth of radix bupleuri root system and improving radix bupleuri quality.

[0010] Further, the promoting growth of radix bupleuri root system includes at least one of 1) to 5):

[0011] 1) Promotes the growth of the above-ground parts of Bupleurum seedlings;

[0012] 2) Promotes the growth of the underground root system of Bupleurum seedlings;

[0013] 3) Promotes the thickening of the taproot of Bupleurum seedlings;

[0014] 4) Increase the fresh weight and / or dry weight of the whole Bupleurum plant;

[0015] 5) Increase the fresh weight / dry weight of Bupleurum root (the part used in medicine).

[0016] Furthermore, the promotion of Bupleurum root growth is achieved through phosphorus solubilization, potassium solubilization, and nitrogen fixation by Ravenella maculatae XS-11.

[0017] Furthermore, improving the quality of Bupleurum includes increasing the content of the main active ingredients in Bupleurum root that exert its medicinal effects: saponins a, b2, d, and f.

[0018] Preferably, the method of application includes root irrigation.

[0019] The application of *Laenia maculata* XS-11 in promoting root growth in *Bupleurum chinense* includes the following process:

[0020] 1) Co-culture of XS-11 and Bupleurum chinense

[0021] Bupleurum seedling cultivation: Select healthy and plump Bupleurum seeds, sow them in sterilized soil after ultrasonic treatment for 20 minutes, water them thoroughly with sterile water, and place them in a greenhouse for cultivation for one month, watering them with Hogland nutrient solution once a week during this period.

[0022] Preparation of bacterial agent: Strawberry strain XS-11 was taken out at -80 ℃. For activation, a small amount of bacterial solution was streaked onto LB solid medium using an inoculation needle in a clean bench (continuous streak method). It was then incubated in the dark at 28 ℃ for 1 day until a single colony appeared. A single colony was picked and aseptically transferred to 1 mL of LB liquid medium, and incubated at 28 ℃ and 180 r / min for 12-16 h until the medium became turbid. 100 µL of the above bacterial solution was inoculated into 20 mL of LB liquid medium and incubated at 28 ℃ and 180 r / min until a suitable absorbance value was reached. The absorbance was measured using a UV spectrophotometer.

[0023] Selecting the culture of one month and the same growth condition of Bupleurum chinense seedlings, adjusting the concentration (OD600) of the bacterial liquid to 0.4, centrifuging the bacterial body to precipitate, resuspending the bacterial body with sterile water, and irrigating the roots of the Bupleurum chinense seedlings, the control group is irrigated with the same volume of sterile water; after one month of co-culture, the Bupleurum chinense seedlings are observed; it is found that the above-ground part and the underground part of the treatment group have different degrees of increase compared with the control group, and the main root is more obvious.

[0024] 2) Comparison of active ingredient content between the XS-11 treatment group and the control group

[0025] The change of the active ingredient content after the XS-11 treatment is tested by HPLC chromatography;

[0026] Preparation of the control and test product solutions: The Chinese Pharmacopoeia takes saikosaponin a and saikosaponin d as the control, and the experiment further increases saikosaponin b2, d and f as the main active ingredients of Bupleurum chinense for testing on the basis of the Pharmacopoeia;

[0027] The experimental steps are as follows: the saikosaponin a, b2, c, d and f control are taken in an appropriate amount, accurately weighed, dissolved in methanol to prepare mixed control stock solutions with mass concentrations of 1.760, 0.02490, 0.4990, 1.990, 0.2505 and 0.4980 mg / mL respectively. About 0.5 g of the Bupleurum chinense sample powder obtained by crushing through a No. 4 sieve is accurately weighed, placed in a conical flask with a plug, 25 mL of 5% ammonia water in methanol solution is added, ultrasonic treatment is carried out at 30 ℃ water temperature for 30 min, filtration is carried out, the container and the drug residue are washed with 10 mL of methanol for 2 times, the washing liquid and the filtrate are combined, and the solvent is recovered. The residue is dissolved in methanol and transferred to a 10 mL volumetric flask, methanol is added for constant volume, shaken well, filtered through a 0.22 μm microporous organic filter membrane, the filtrate is taken, i.e. the test sample, HPLC chromatography test is carried out, and the results show that the saikosaponin a, b2, d and f of the treatment group are significantly higher than those of the control group.

[0028] Preferably, the HPLC chromatography conditions in 2) are as follows:

[0029] Column: Agela Venusil MPC (4.6 mm x 250 mm, 5 μm); mobile phase: A (water) - B (acetonitrile) gradient elution, program: 0-10 min, 34% B; 10-42 min, 34%-50% B; flow rate: 1.0 m / min, column temperature: 30 ℃, detection wavelength: 210 nm and 254 nm, injection volume: 10 μL.

[0030] In order to further illustrate the present application, the extraction, culture and identification process of the aforementioned Acerola bacteria XS-11 are described in detail below, which specifically includes the following steps:

[0031] S1, Bupleurum root rhizosphere soil collection process

[0032] Take the root system soil, shake off the plant root peripheral soil, brush the soil attached to the root surface with a brush, remove impurities with a 20-mesh sieve, and store in a sterile bag in a -80 ℃ refrigerator for standby;

[0033] S2, Bupleurum root rhizosphere bacteria isolation process

[0034] The soil supernatant with a concentration of 10 -2 g / ml is sequentially gradient-diluted to a concentration of 10 -3 , 10 -4 , and 10 -5 g / ml. 100 μL of the soil suspension with a concentration of 10 -3 , 10 -4 , and 10 -5 g / ml is uniformly coated on LB solid culture medium, and 3 repeated samples are prepared for each concentration.

[0035] The culture is placed in a 28 ℃ constant temperature incubator for inverted culture for 1-2 days. The number of bacterial colonies growing on each culture medium is recorded, and colonies with significant differences in size and shape are picked and inoculated on new LB plates. Three generations are purified by the plate streaking method.

[0036] The purified strain is inoculated into LB liquid medium, and cultured overnight at 28 ℃ and a rotation speed of 180 r / min until the OD (600 nm) is between 0.2 and 0.4. The bacterial liquid is added to LB liquid medium containing 20% glycerol, frozen in liquid nitrogen, and stored in a -80 ℃ refrigerator for standby.

[0037] S3, Rhizosphere bacterial morphology and molecular identification

[0038] (1) Morphological identification

[0039] The bacteria are inoculated into LB medium and cultured in a 28 ℃ constant temperature incubator for 12-24 h. After the colonies grow, their morphology, size, color, and other significant characteristics are observed, and single colonies are picked for microscopic examination. The bacterial morphology is observed under a microscope, and Gram staining is used to determine that the bacteria are negative.

[0040] (2) Molecular identification

[0041] The strain stored in the -80 ℃ refrigerator is inoculated into LB solid culture medium, and single colonies are picked and cultured in LB liquid medium at a rotation speed of 180 r / min and 28 ℃ for 12 h to obtain a bacterial liquid.

[0042] The DNA of the strain was extracted by using the Cisco bacterial genome DNA rapid extraction kit, the DNA concentration and quality were detected, and the extracted DNA was immediately subjected to the next step test or stored in a-20℃ refrigerator;

[0043] The extracted DNA was used as a template to configure a 20 µL PCR reaction system, 1 uL of each of bacterial universal primers 27f (gene sequence table 2: 5'-AGAGTTTGATCCTGGCTCAG-3) and 1492r (gene sequence table 3: 5'-TTAAGGTGATCCAGCCGCA-3), 2 µL of DNA template, 6 µL of ddH2O, and 10 µL of 2×Rapid Taq Master Mix; the mixture was thoroughly mixed and placed in a PCR instrument for 16S rRNA gene fragment amplification, and the bacterial liquid was identified as Rahnella aceri XS-11.

[0044] Preferably, the root system soil in S1 is sampled by a five-point sampling method, and the root system soil located at 0-20 cm of the plant is sampled, and the rhizosphere soil sample of Bupleurum is derived from a Bupleurum planting base in the Xishan of Baoji City, Shaanxi Province (north latitude 34°15'-34°38', east longitude 106°36'-107°05').

[0045] Preferably, the preparation process of the soil supernatant in S2 is as follows: 1 g of the rhizosphere soil sample is weighed and placed in a triangular flask containing 99 mL of sterile water, and the flask is placed in a 28 ℃, 180 r / min shaking culture for 30 min.

[0046] Preferably, the gram staining in S3 includes the following steps:

[0047] ①A drop of distilled water is dropped in the center of the glass slide, a single colony is picked up with an inoculation ring and mixed with the drop of water on the glass slide, an outer flame of an alcohol lamp is used to evaporate the water, and the bacteria are fixed on the glass slide;

[0048] ②Oxalic acid amine crystal violet dye is added dropwise, and the staining is performed for 1 min, and then washed with water;

[0049] ③Gram iodine solution is added dropwise for 1 min, and then washed with water;

[0050] ④95% ethanol is used for decolorization for about 20 s, and then washed with water;

[0051] ⑤Ponceau dye is added dropwise for re-staining for 2 min, and then washed with water, and then naturally dried for microscopic examination.

[0052] Pre-denaturation at 94 ℃ for 3 min, denaturation at 94 ℃ for 5 s, annealing at 52 ℃ for 15 s, extension at 72 ℃ for 30 s, for a total of 35 cycles, and finally extension at 72 ℃ for 10 min. The PCR product was sent to Shenguo Bioengineering (Shanghai) Co., Ltd. for sequencing. The NCBI database was used for homology comparison, and the top 10 sequences were screened out. The 16S rRNA sequence was downloaded, and the phylogenetic tree was constructed using software MEGA11.0, as shown in FIG. 1. According to the gene sequence table 1, it was identified that the strain XS-11 was Rahnella aceris. Figure 2

[0053] Preferably, the biological activity screening process of Rahnella aceris XS-11 is as follows:

[0054] Ashby nitrogen-free medium: mannitol 10 g, KH2PO4 0.2 g, MgSO4·7H2O 0.2 g, NaCl 0.2 g, CaCO3 5 g, agar powder 15-20 g, pH=6.8-7.0;

[0055] PKO organic phosphorus medium: glucose 10 g, (NH4)2SO4 0.5 g, NaCl 0.3 g, KCl 0.3 g, MgSO4·7H2O 0.3 g, CaCO3 5 g, FeCl3 0.03 g, egg yolk liquid (lecithin source) 5 mL, pH=7.0;

[0056] Potassium-lysing medium: sucrose 5.0 g, magnesium sulfate 0.5 g, disodium hydrogen phosphate 2.0 g, ferric chloride 0.005 g, calcium carbonate 0.1 g, soil minerals 1.0 g, agar 18.0 g / L, pH=7.2;

[0057] The biological functions of the tested Rahnella aceris XS-11 include:

[0058] Phosphorus solubilization: the strain was inoculated in the PKO medium and cultured in a constant temperature incubator at 28 ℃ for 4-7 d, and whether transparent circles were generated was observed.

[0059] Potassium-lysing: the strain was inoculated in the potassium-lysing medium and cultured in a constant temperature incubator at 28 ℃ for 4-7 d, and whether transparent oil droplet-shaped colonies and mucous edge expansion were formed on the medium was observed.

[0060] Nitrogen fixation: the strain was inoculated in the Ashby nitrogen-free medium and cultured in a constant temperature incubator at 28 ℃ for 4-7 d, and whether the colonies with gelatinous, translucent or milky white, surface wet and mucous layer were formed on the medium was observed.

[0061] ​Compared with the prior art, the present application has the beneficial effects that:

[0062] 1. The present application isolates and obtains Rahnella aceris XS-11 from the rhizosphere soil of the native producing area (Baoji, Shaanxi) of Bupleurum, which has natural adaptability to the growth environment of Bupleurum and is easy to colonize the root system; through function verification such as phosphorus release (PKO culture medium), potassium release (potassium release culture medium), and nitrogen fixation (Ashby culture medium), XS-11 can simultaneously activate soil insoluble phosphorus and potassium and fix nitrogen, providing balanced nutrition for Bupleurum and reducing the dependence on chemical fertilizers; co-culture experiments show that the aboveground biomass of Bupleurum in the XS-11 treatment group increases by 28%, the root length increases by 35%, and the taproot development is more robust, shortening the planting cycle; HPLC detection confirms that the contents of saikosaponin a, b2, d, and f in the XS-11 treatment group are significantly higher than those in the control group, and the increase of saikosaponin a and d (core indicator components of the pharmacopoeia) is more than 40%, directly improving the medicinal value.

[0063] 2. The present application realizes precise identification of the strain by combining morphological, Gram staining and 16S rRNA phylogenetic analysis; establishes a standardized process of gradient dilution-plate streaking-glycerol preservation to ensure the reliability of strain isolation, purification and activity.

[0064] 3. The bacterial agent of the present application is simple to prepare (LB liquid expansion culture + root irrigation), low in cost, and strong in strain specificity, which can replace part of the chemical fertilizer, reduce non-point source pollution, provide core technical support for "quality improvement and efficiency increase" of Bupleurum native medicinal materials, and promote green cultivation of Chinese herbal medicines. BRIEF DESCRIPTION OF DRAWINGS

[0065] Figure 1 Fig. 1 is a morphological identification result of XS-11 of the present application, wherein Figure 1 a is the morphology of strain XS-11 on the front of the LB plate, and b is the morphology of strain XS-11 on the back of the LB plate; c is the Gram staining result of strain XS-11;

[0066] Figure 2 Fig. 2 is a gene phylogenetic tree of XS-11 of the present application;

[0067] Figure 3 Fig. 3 is the bacterial biological culture phenomenon of the culture medium of XS-11 of the present application, wherein Figure 3 a shows that XS-11 produces a transparent circle in the PKO culture medium; Figure 3 b shows that XS-11 appears as oil droplets in the potassium release culture medium, Figure 3 c shows that XS-11 appears as transparent mucus in the Ashby nitrogen-free culture medium;

[0068] Figure 4 Fig. 4 is the growth promotion effect of co-culture of XS-11 of the present application and Bupleurum, whereinFigure 4 a is a blank control group, Figure 4 b is an effect diagram of using XS-11 to co-culture with Bupleurum;

[0069] Figure 5 HPLC results of the active ingredient content comparison between the XS-11 treatment group and the control group of the present application. DETAILED DESCRIPTION

[0070] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the prior known technology. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments.

[0071] Embodiment 1:

[0072] 1. Bupleurum rhizosphere soil collection process

[0073] The Bupleurum rhizosphere soil sample was derived from the Bupleurum planted base in the Xishan of Baoji City, Shaanxi Province (north latitude 34°15'-34°38', east longitude 106°36'-107°05'), and the root system soil at 0-20 cm of the plant was taken by the five-point sampling method. The peripheral soil of the plant root system was shaken off, the soil attached to the root surface was brushed with a brush, 20-mesh sieve was used to remove impurities, and the soil was put into a sterile bag and stored in a-80 ℃ refrigerator for standby.

[0074] 2. Bupleurum rhizosphere bacteria separation process

[0075] 1 g of the rhizosphere soil sample was weighed and put into a triangular flask containing 99 mL of sterile water, and was placed in a 28 ℃, 180 r / min speed shaking culture for 30 min. The soil supernatant with a concentration of 10 -2 g / ml was obtained, and the soil suspension with a concentration of 10 -3 , 10 -4 , 10 -5 was gradiently diluted in turn. 100 μL of the soil suspension with a concentration of 10 -3 , 10 -4 , 10 -5 was taken, and was uniformly coated on LB solid culture medium, with 3 repeats for each concentration. It was placed in a 28 ℃ constant temperature incubator and cultured for 1-2 d. The number of bacterial colonies growing on each culture medium was recorded, and the colonies with large size difference in morphology were picked and inoculated on new LB plates. The 3rd generation was purified by plate streaking method. The purified strain was inoculated into LB liquid medium, and was cultured at 28 ℃, 180 r / min speed overnight until the OD (600 nm) was between 0.2-0.4. The bacterial liquid was taken and added into LB liquid medium containing 20% glycerol. After liquid nitrogen quick freezing, it was stored in a-80 ℃ refrigerator for standby.

[0076] 3. Rhizosphere bacterial morphological and molecular identification process

[0077] (1) Morphological identification

[0078] Inoculate the bacteria into LB medium and place in a 28 °C constant temperature incubator for 12-24 h. After the colonies grow, observe their morphology, size, color and other significant characteristics, pick single colonies for slide preparation and microscopic examination, and observe the bacterial morphology under a microscope. Gram staining is used to determine whether it is a negative bacterium.

[0079] Gram staining:

[0080] ①Drop a drop of distilled water in the center of the slide, pick up a single colony with a inoculation ring and mix it with the water drop on the slide, use the outer flame of an alcohol lamp to evaporate it, and fix the bacterial body on the slide;

[0081] ②Add oxalic acid amine crystal violet dye, stain for 1 min, and wash with water;

[0082] ③After staining with Gram iodine solution for 1 min, wash with water;

[0083] ④Decolorize with 95% ethanol for about 20 s, and wash with water;

[0084] ⑤Add safranin dye for restaining for 2 min, wash with water, and examine under a microscope after natural drying. The results are shown in Figure 1 .

[0085] (2) Molecular identification

[0086] Take out the strain stored in the -80 °C refrigerator, inoculate it into LB solid medium, pick a single colony on LB liquid medium, and shake culture at 180 r / min and 28 °C for 12 h to obtain a bacterial solution. Use the Scikey bacterial genome DNA rapid extraction kit to extract the strain DNA, detect the DNA concentration and quality, and immediately perform the next step of the experiment or store it in the -20 °C refrigerator.

[0087] Use the extracted DNA as a template to configure a 20 µL PCR reaction system, 1 µL each of bacterial universal primers 27f (gene sequence Table 2: 5'-AGAGTTTGATCCTGGCTCAG-3) and 1492r (gene sequence Table 3: 5'-TTAAGGTGATCCAGCCGCA-3), 2 µL of DNA template, 6 µL of ddH2O, and 10 µL of 2×Rapid Taq Master Mix. Mix well and put into a PCR instrument to amplify the 16S rRNA gene fragment.

[0088] PCR procedure: 94 ℃ pre-denaturation for 3 min, 94 ℃ denaturation for 5 s, 52 ℃ annealing for 15 s, 72 ℃ extension for 30 s, a total of 35 cycles, and finally 72 ℃ extension for 10 min. The PCR product was sent to Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing. The homology comparison was performed by using the NCBI database, and the top 10 sequences with the highest scores were screened out. The 16S rRNA sequences were downloaded, and the phylogenetic tree was constructed by using software MEGA11.0, as shown in Figure 2 According to the gene sequence table 1, it is identified that the strain XS-11 is Rahnella aceris.

[0089] 4. XS-11 biological activity screening:

[0090] Ashby nitrogen-free medium: mannitol 10 g, KH2PO4 0.2 g, MgSO4·7H2O 0.2 g, NaCl 0.2 g, CaCO3 5 g, agar powder 15-20 g, pH = 6.8-7.0;

[0091] PKO organic phosphorus medium: glucose 10 g, (NH4)2SO4 0.5 g, NaCl 0.3 g, KCl 0.3 g, MgSO4·7H2O 0.3 g, CaCO3 5 g, FeCl3 0.03 g, egg yolk liquid (lecithin source) 5 mL, pH = 7.0;

[0092] Potassium-lysing medium: sucrose 5.0 g, magnesium sulfate 0.5 g, disodium hydrogen phosphate 2.0 g, ferric chloride 0.005 g, calcium carbonate 0.1 g, soil mineral 1.0 g, agar 18.0 g / L, pH = 7.2;

[0093] Bacterial biological function exploration, as shown in Figure 3

[0094] Phosphorus-lysing: the strain was inoculated in the PKO medium, and cultured in a constant temperature incubator at 28 ℃ for 4-7 days. Whether transparent circle was produced was observed.

[0095] Potassium-lysing: the strain was inoculated in the potassium-lysing medium, and cultured in a constant temperature incubator at 28 ℃ for 4-7 days. Whether oil droplet-like colonies were formed on the medium and the edge was mucous expansion was observed.

[0096] Nitrogen-fixing: the strain was inoculated in the Ashby nitrogen-free medium, and cultured in a constant temperature incubator at 28 ℃ for 4-7 days. Whether the colonies with gelatinous, translucent or milky white, surface wet and mucous layer were formed on the medium was observed.

[0097] 5. XS-11 effect test process on bupleurum promotion: ​

[0098] (1) XS-11 and Bupleurum co-culture

[0099] Bupleurum seedling culture: Select healthy and full Bupleurum seeds, after ultrasonic treatment for 20 min, spread them in sterilized soil, pour sterile water to soak, and then place them in a greenhouse for one month. During this period, pour Hoagland nutrient solution every week.

[0100] Preparation of bacterial agent: Take strain XS-11 at -80 ℃. When activated, use a inoculation needle to draw a line on LB solid medium with a small amount of bacterial liquid (continuous streaking method) in a clean bench, and place it in a 28 ℃ constant temperature incubator for dark culture for 1 day until single colonies appear. Pick single colonies and transfer them to 1 mL of LB liquid medium under sterile conditions, and culture at 28 ℃, 180 r / min for 12-16 h until the medium becomes turbid. Take 100 μL of the above bacterial liquid and inoculate it into 20 mL of LB liquid medium, and culture at 28 ℃, 180 r / min until the appropriate absorbance value is reached. Measure it using a UV spectrophotometer.

[0101] Select Bupleurum seedlings that have been cultured for one month and have the same growth conditions, adjust the bacterial liquid concentration (OD600) to 0.4, centrifuge the bacteria to precipitate, resuspend the bacteria with sterile water, and perform root irrigation treatment on the Bupleurum seedlings. The control group is irrigated with the same volume of sterile water. After co-culturing for one month, the Bupleurum seedlings are observed for their properties.

[0102] According to the results shown in Figure 4 , it was found that the aboveground and underground parts of the treatment group increased to varying degrees compared with the control group, and the main roots were more obvious.

[0103] (2) Comparison of active ingredient content between XS-11 treatment group and control group

[0104] HPLC chromatographic conditions:

[0105] Chromatographic column: Agela Venusil MPC (4.6 mm x 250 mm, 5 μm); mobile phase: A (water) - B (acetonitrile) gradient elution, program: 0-10 min, 34% B; 10-42 min, 34%-50% B; flow rate: 1.0 m / min, column temperature: 30 ℃, detection wavelength: 210 nm and 254 nm, injection volume: 10 μL.

[0106] The experimental procedure is as follows: Accurately weigh appropriate amounts of saikosaponins a, b2, c, d, and f, respectively, and dissolve them in methanol to prepare mixed reference stock solutions with mass concentrations of 1.760, 0.02490, 0.4990, 1.990, 0.2505, and 0.4980 mg / mL, respectively. Accurately weigh approximately 0.5 g of powder obtained from pulverizing the Bupleurum sample (passing through a No. 4 sieve), place it in a stoppered conical flask, add 25 mL of methanol solution containing 5% ammonia, sonicate at 30 ℃ for 30 min, filter, and wash the container and residue twice with 10 mL of methanol. Combine the washings and filtrate, and recover the solvent. Dissolve the residue in methanol and transfer it to a 10 mL volumetric flask, dilute to volume with methanol, shake well, and filter through a 0.22 μm microporous organic filter membrane. Collect the filtrate to obtain the final product.

[0107] according to Figure 5 As shown in the figure, the results showed that the levels of saikosaponins a, b2, d, and f in the treatment group were significantly higher than those in the control group.

[0108] Comparative Example 1:

[0109] Preparation of reference and test solutions: The Chinese Pharmacopoeia uses saikosaponin a and saikosaponin d as reference standards. In this experiment, the main active ingredients of Bupleurum chinense, saikosaponin b2, d and f, are added as test components in addition to the reference standards.

[0110] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. Application of Rahnella ceris XS-11 in promoting growth of Bupleurum falcatum, characterized in that, The classification name of the Ralstonia insidiosa XS-11 is Ralstonia insidiosa, which is preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, with the preservation number CGMCC No. 35248 and the preservation date July 16, 2025; The application refers to applying Ralstonia insidiosa XS-11 or a microbial agent containing Ralstonia insidiosa XS-11 to Bupleurum chinense seedlings. 2.The application of Ralstonia pickettii XS-11 in promoting the growth of Bupleurum according to claim 1, characterized in that, The microbial agent refers to a bacterial suspension containing Ralstonia insidiosa XS-11 with a dosage of OD600 = 0.

4.

3. The use of Ralstonia pickettii XS-11 in promoting the growth of Bupleurum according to claim 1, characterized in that, The application of the Ralstonia insidiosa XS-11 or the microbial agent in promoting the growth of Bupleurum chinense root system and improving the quality of Bupleurum chinense.

4. The use of Ralstonia pickettii XS-11 in promoting the growth of Bupleurum according to claim 3, characterized in that, The promotion of the growth of Bupleurum chinense root system includes at least one of 1) to 5): 1) promoting the growth of the aboveground part of Bupleurum chinense seedlings; 2) promoting the length growth of the root system of the underground part of Bupleurum chinense seedlings; 3) promoting the thickening of the main root of the root system of Bupleurum chinense seedlings; 4) increasing the fresh weight and / or dry weight of the whole plant of Bupleurum chinense; 5) increasing the fresh weight and / or dry weight of the root system (medicinal part) of Bupleurum chinense.

5. The use of Ralstonia pickettii XS-11 in promoting the growth of Bupleurum according to claim 3, characterized in that, The promotion of the growth of Bupleurum chinense root system is achieved by the phosphorus and potassium solubilization and nitrogen fixation of Ralstonia insidiosa XS-11.

6. The use of Ralstonia pickettii XS-11 in promoting the growth of Bupleurum according to claim 3, characterized in that, The improvement of the quality of Bupleurum chinense includes increasing the content of the main active ingredients of Bupleurum chinense in the root system, i.e., saponins a, b2, d, and f.

7. The use of Ralstonia pickettii XS-11 in promoting the growth of Bupleurum according to claim 1, characterized in that, The method of application includes root irrigation.

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