An endophytic bacterium that induces ginseng to accumulate saponins and its application

Abstract

The present invention discloses an endophytic bacterium that induces ginseng to accumulate saponin components and its application. A strain of Mesorhizobium sp. G198 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration Committee, with a deposit number of CGMCC No: 30089 and a deposit date of March 21, 2024. The G198 strain of the present invention is a new species, and it is reported for the first time that it has the activity of inducing the accumulation of saponin content. G198 can promote the accumulation of total saponin content in ginseng adventitious roots and potted ginseng, and specifically promotes the accumulation of F and Rh family saponin content, and is expected to be used to develop functional microbial fertilizers that can increase the content of ginsenosides and improve the quality of ginseng.

Description

Technical Field

[0001] The invention belongs to the field of agricultural microorganisms and relates to an endophytic bacterium for inducing ginseng to accumulate saponin components and application thereof. Background Art

[0002] Ginseng (Panaxginseng CA Meyer) is a precious Chinese medicinal material with a wide range of medicinal activities. Its main active ingredient is the secondary metabolite triterpenoid ginsenosides, which have clear therapeutic effects in improving immunity, anti-tumor and preventing cardiovascular diseases. According to different ginseng planting methods, ginseng is divided into wild-cultivated forest ginseng and garden ginseng cultivated in farmland. The two types of ginseng have large differences in the content of active substances (ginsenosides), especially the forest ginseng with a long growth period has a higher content of saponins, and the medicinal effects of ginseng are more significant. In addition, our preliminary research shows that the saponin content has a cumulative effect year by year. However, the current forest ginseng resources are limited, and the output is far from meeting the market. Therefore, increasing the saponin content of garden ginseng and ensuring the quality of garden ginseng are important guarantees for the healthy development of the cultivated ginseng industry in the future.

[0003] In recent years, many cutting-edge studies have shown that the accumulation of plant secondary metabolites is closely related to the associated root microorganisms. Among them, the key bacteria in the rhizosphere of Astragalus membranaceus are significantly related to the content of astragaloside and calycosin; the rhizosphere microorganisms of corn and Arabidopsis thaliana have been shown to be involved in regulating the synthesis of triterpenes and flavonoids in plants. Based on this, we speculate that the root microorganisms associated with ginseng may be involved in regulating the synthesis of ginsenosides (triterpenoid compounds). Therefore, the exploration and development of functional microorganisms that promote saponin synthesis will provide theoretical support for the further development and promotion of functional microbial products that can improve the quality of ginseng. At present, most of the research on Mesorhizobium microorganisms focuses on microbial nitrogen fixation and plant growth promotion. However, there is no specific report on the promotion of ginseng saponin synthesis by Mesorhizobium microorganisms. Summary of the invention

[0004] The object of the present invention is to provide a strain of Mesorhizobium sp. G198 in view of the above-mentioned deficiencies in the prior art.

[0005] Another object of the present invention is to provide the application of Mesorhizobium sp. G198.

[0006] Another object of the present invention is to provide a method for increasing the content of saponins in ginseng adventitious roots and potted ginseng.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] A strain of Mesorhizobium sp. G198 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No: 30089, and the deposit date is March 21, 2024.

[0009] The Mesorhizobium G198 bacterial agent of the present invention.

[0010] The preparation method of the mesorhizobium G198 bacterial agent of the present invention comprises inoculating the mesorhizobium G198 with a deposit number of CGMCC No: 30089 into a 1 / 2 TSB liquid culture medium and culturing the culture medium until the strain concentration is 1*10 8 CFU / mL, the bacterial solution is centrifuged, repeatedly eluted with sterile distilled water, and centrifuged to obtain clean bacteria without culture medium components, and finally sterile distilled water is added to resuspend to obtain the bacterial agent; wherein the culture conditions are: temperature 25-30°C, rotation speed 100-150 rpm, and dark culture for 70-75h.

[0011] As a preferred embodiment of the present invention, the culture conditions are: temperature 28° C., rotation speed 120 rpm, and dark culture for 72 hours.

[0012] The use of the mesorhizobium G198 or the mesorhizobium G198 agent in promoting the accumulation of saponin components in ginseng or in preparing a product that promotes the accumulation of saponin components in ginseng.

[0013] As a preferred embodiment of the present invention, the ginsenoside is one or more of Rg1, Re, Rc, Ro, Rb1, Rd, Rh1, R1, F2, 20DRf, Rh2, Rh4 and CK.

[0014] As a preferred embodiment of the present invention, the mesorhizobium G198 or the bacterial agent of the mesorhizobium G198 is used to increase the content of Rh1, Rh2, Rh4 (Rh family), F2 and CK saponins in ginseng adventitious roots.

[0015] As a preferred embodiment of the present invention, the use of Mesorhizobium G198 or the bacterial agent of Mesorhizobium G198 is used to increase the content of Rg1, Re, Rc, Ro, Rb1, Rd, Rh1, R1, F2 and 20DRf saponins in potted ginseng.

[0016] A method for increasing the content of saponin components in ginseng adventitious roots, wherein the Mesorhizobium sp. G198 is prepared at a final concentration of 1*10 7CFU / mL is inoculated into the ginseng adventitious root liquid culture system that has been cultured for 18-22 days, and co-cultured for 10 days or more; the co-culture conditions are temperature 25-30℃, rotation speed 100-150 rpm, dark culture; preferably temperature 28℃, rotation speed 120 rpm, dark culture. The ginseng adventitious root culture liquid system is a 50mL culture bottle, in which 30mL of adventitious root culture medium is inoculated with 1g of fresh weight ginseng adventitious root liquid culture for 18-22 days or cultured until the ginseng adventitious root biomass reaches 2.5-3.5g of fresh weight. The adventitious root culture medium formula is as follows: 30g sucrose, 5mg IBA hormone, 3.35g MS powder, 1L double distilled water, pH=6.0; the liquid culture conditions are 25℃ constant temperature dark culture, rotation speed 120 rpm.

[0017] A method for increasing the content of saponin components in potted ginseng, comprising applying the clean bacterial solution of G198 to the rhizosphere soil of ginseng seedlings, 50 mL of bacterial solution per pot each time, and ensuring that the concentration of the bacterial solution in the inoculated soil is 1*10 7 CFU / g soil (i.e. the microbial content per gram of soil is 1*10 7 CFU).

[0018] Beneficial effects:

[0019] The strain G198 of the present invention is identified as a new species (Mesorhizobium sp.) of Mesorhizobium (Mesorhizobium sp.), and the present invention reports for the first time that the strain has the activity of inducing the accumulation of saponin content. G198 can promote the accumulation of total saponin content in ginseng adventitious roots and potted ginseng, specifically promotes the accumulation of saponin content of F and Rh families, and is expected to be used in the development of functional microbial fertilizers that can increase the content of ginsenosides and improve the quality of ginseng. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Results of constructing the phylogenetic tree of G198 microorganisms

[0021] Figure 2 G198 Microbial Morphological Observation

[0022] Figure a shows the colony morphology of G198, and Figure b shows the bacterial morphology of G198

[0023] Figure 3 Co-culture of Ginseng Adventitious Roots and Strain G198

[0024] Biomaterial Deposit Information

[0025] G198, classified and named Mesorhizobium sp., is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC No: 30089, and the deposit date is March 21, 2024. DETAILED DESCRIPTION

[0026] Example 1

[0027] 1. Description of microbial culture

[0028] The G198 strain was obtained in September 2021 by screening the fibrous roots of 21-year-old ginseng at the ginseng planting base in Diaoyu Village, Huadian Town, Huanren County, Liaoning Province (125°47′E, ​​41°15′N).

[0029] 2. Molecular Biology Identification

[0030] The full length of the 16S rDNA sequence of the G198 strain is 1423bp, as shown in SEQ ID NO.1. The sequence was submitted to the NCBI platform GenBank for BLAST similarity comparison. The results showed that the strain had a high homology with some strains of the genus Mesorhizobium. Strains with a similarity of more than 97% to the target sequence were selected, and the phylogenetic tree was constructed using the Neighbor-Joining Algorithm (NJ) method using ClustalX and Mega5.0 software. The results showed that the G198 strain was not classified as a small branch with any homologous microorganisms, and it was preliminarily speculated that the microorganism was a new species of Mesorhizobium (Mesorhizobium.Sp.) ( Figure 1 ).

[0031] 3. Morphological identification

[0032] The morphological observation of G198 strain after growing on 1 / 2TSB solid medium for 72 hours showed that the colonies were typical yellow-white opaque, with a slight bulge in the middle, relatively sticky, generally smooth, shiny, and neatly edged ( Figure 2 Figure a). Microscopic observation showed that the Gram stain was red, Gram-negative, the length of the bacteria was about 7-10 μm, and the bacteria were rod-shaped ( Figure 2 (Figure b), which is consistent with the microbial characteristics of Mesorhizobium.

[0033] 4. Physiological and biochemical identification

[0034] The results of physiological and biochemical indexes of G198 strain are shown in Table 1. The indexes include: BTB culture alkali production, litmus milk reaction, maltose carbon source utilization, L-arabinose carbon source utilization, sucrose carbon source utilization and other indicators are positive; sensitive to antibiotics such as chloramphenicol (50μg / mL), gentamicin (5μg / mL), tetracycline (10μg / mL) and kanamycin (50μg / mL); 3-ketolactose reaction, high salt tolerance (5%), pH = 4.5 survival, pH = 10 survival, indole test, gelatin liquefaction and other indicators are negative. By consulting the Bergey's Bacteria Identification Manual and the Common Bacteria System Identification Manual, it was preliminarily determined that the G198 strain meets the physiological and biochemical characteristics of Mesorhizobium microorganisms.

[0035] Table 1. Results of physiological and biochemical index tests on G198 strain

[0036]

[0037]

[0038] In summary, strain G198 was named Mesorhizobium ginsenosides.

[0039] 5. G198 culture conditions

[0040] Preparation of 1 / 2TSB medium: 15g TSB (oxoid, CM0129), 1L distilled water, 20g agar powder, pH = 6.5, sterilize at 121℃ for 25min, prepare solid slant medium, and do not add agar powder to make 1 / 2TSB liquid medium. 1 / 2TSB solid medium is cultured at a constant temperature of 28℃; 1 / 2TSB liquid medium is cultured at a constant temperature of 28℃ and a rotation speed of 120 rpm.

[0041] 6. Storage status

[0042] G198 has been deposited in the General Microbiology Center of China Culture Collection Administration, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The strain number is G198; the deposit number is CGMCCNo:30089, and the deposit date is March 21, 2024.

[0043] Example 2 G198 bacteria promote the accumulation of saponin content in ginseng adventitious roots

[0044] (1) Ginseng adventitious root culture

[0045] The ginseng adventitious root culture liquid system is a 50mL culture bottle, which contains 30mL of culture medium. The formula of the adventitious root culture medium is as follows: 30g sucrose, 5mg IBA hormone (PhytoTech1460, CAS: 133-32-4), 3.35g MS powder (PhytoTech1962, CAS: M519), 1L double distilled water, pH = 6.0. 1g fresh weight of ginseng adventitious root is inoculated, and the liquid culture conditions are 25°C constant temperature dark culture and 120 rpm. When the ginseng adventitious root grows to 20 days (biomass is 2.5-3.5g fresh weight), G198 bacteria are ready for inoculation.

[0046] (2) Preparation of G198 cells

[0047] The solid culture block of G198 was selected and inoculated into 1 / 2TSB liquid medium (100mL 1 / 2TSB liquid medium, 250mL Erlenmeyer flask) for bacterial culture. The conditions for bacterial culture were: temperature 28°C, speed 120 rpm, dark culture for 72h, and the strain was cultured to 1*10 8 CFU / mL, and obtain the test bacterial solution. The bacterial solution was centrifuged in a centrifuge (3500 rpm, room temperature) for 15 minutes, and the supernatant was removed to obtain the bacterial precipitate. The bacterial precipitate was repeatedly eluted with sterile distilled water and centrifuged twice (same conditions as above) to obtain clean bacteria without culture medium components. Finally, sterile distilled water was used as the solvent to adjust the bacterial concentration to 1*10 8 CFU / mL is reserved.

[0048] (3) Liquid co-culture of G198 strain and ginseng adventitious roots

[0049] Clean bacteria according to the final concentration of 1*10 7 CFU / mL inoculated into ginseng adventitious roots (20 days) liquid culture system ( Figure 3 ), and the content of various saponins in adventitious roots was detected after co-cultivation for 10 days under the conditions of temperature 28°C, rotation speed 120 rpm, and dark culture. First, the adventitious roots were taken out, and the adventitious roots were repeatedly rinsed 3 times with distilled water, and then the surface moisture of the roots was dried with absorbent paper, placed in a 5mL centrifuge tube and dried in an oven at 40°C for 24h for subsequent saponin content detection. The dried adventitious roots were collected in a 2ml centrifuge tube, and after adding zirconium oxide beads, they were ground into powder by a tissue crusher (60HZ, 2min) for later saponin content detection.

[0050] Weigh 30 mg of adventitious root powder accurately into a new 2 ml centrifuge tube, add 1.5 mL of 70% methanol solution (methanol: water = 7:3, v / v), shake and mix thoroughly, let stand for 12 hours, and then ultrasonically extract for 50 minutes (power 500W, frequency 40kHZ) to obtain the extraction solution. Then centrifuge (13000r, room temperature) for 10 minutes to obtain the supernatant, use a 0.22μm microporous filter membrane to remove impurities, and obtain the solution to be tested for saponin content.

[0051] The content of various saponins in adventitious roots was determined by using the UPLC-TQ-MS / MS method. The detection method is as follows: the chromatographic column uses a Waters ACQUITYUPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) column, the column temperature is set to 40°C; the mobile phase is 0.1% formic acid-acetonitrile (A) and 0.1% formic acid-water (B), the flow rate is set to 0.4 mL / min, and the injection volume is 1 μL. The elution gradient was 0-2.5min, 28-31% A; 2.5-3min, 31-35% A; 3-4.5min 35-36% A; 4.5-6min 36-37% A; 6-7min 37-50% A; 7-9min 50-98% A; 9-10min 98-98% A; 10-11min 98-28% A; 11-13min 28% A. Mass spectrometry conditions: In combination with the properties of different saponin compounds, the mass spectrometry was operated in negative ion mode, and the parameters such as Q1 MASS (Da), Q3 MASS (Da), declustering voltage (DP / V), collision energy (CE / eV) and emission voltage (CXP / V) are shown in Table 2 below.

[0052] Table 2. UPLC-TQ-MS / MS mass spectrometry conditions

[0053]

[0054]

[0055] Note: DP: Declustering potential; CE: Collision energy; CXP: Ejection voltage.

[0056] Combined with the ginsenoside content of different samples, saponin standards were used to determine the saponin quantitative standard curve and its applicable range. The specific standard curve is shown in Table 3.

[0057] Table 3. Regression curve of saponin quantitative calculation

[0058]

[0059]

[0060] Note: The concentration unit is μg / mL.

[0061] (4) Results of the total saponin accumulation induced by co-culture of G198 strain and adventitious roots

[0062] The G198 bacterial group had a significant effect on promoting the accumulation of total saponin content in ginseng adventitious roots, which was significantly different from the blank group (P≤0.05, Table 4), and the total saponin content increased by 83.6%. Analysis of the changes in the content of single saponins in the G198 bacterial group showed that after bacterial treatment, the content of saponins Rh1, Rh2, Rh4 (Rh family) and CK increased from zero to one, and the content was significantly different from that in the control group (P≤0.05). In addition, the content of F2 increased significantly, which was significantly different from that in the control group (P≤0.05).

[0063] Table 4. Results of G198 strain promoting the accumulation of saponin content in ginseng adventitious roots

[0064]

[0065] Note: The control group represents the sterile treatment group, Total represents the total saponin content, and different lowercase letters represent significant differences at the 0.05 level.

[0066] Example 3 G198 bacteria promote the accumulation of saponin content in potted ginseng

[0067] (1) Conditions for growing ginseng in pots

[0068] For potted ginseng, choose 3-year-old seedlings, the variety is Erma Ya. For planting, choose flower pots with a diameter of 15 cm and a depth of 12 cm, with one ginseng seedling in each pot. For planting, choose peat soil: vermiculite: perlite = 4:1:1 (volume ratio). The soil moisture is adjusted to a state where it can be squeezed into a ball and loosened when released. Each pot contains 250g of soil matrix. The ginseng culture conditions are: air humidity is controlled at 40-45%, the growth temperature is 25°C, 8 hours of light / 16 hours of darkness, and ginseng seedlings are watered every 7 days, 50mL / pot each time. After the ginseng seedlings have grown for 45 days, inoculate with G198 bacterial solution (the bacterial solution is obtained as above, and the concentration is adjusted to 5*10 7 CFU / mL), 50mL of bacterial solution / pot each time, ensuring that the concentration of inoculated soil bacterial solution is 1g and the amount of soil microorganisms is 1*10 7 CFU. On the 12th day after inoculation of microorganisms, the changes in ginsenoside content were detected.

[0069] First, take out the ginseng roots after microbial induction, rinse them repeatedly with distilled water for 3 times to remove soil impurities, and then use absorbent paper to absorb the moisture on the root surface. Then cut the ginseng roots into 5-10mm fragments and place them in a 5mL centrifuge tube and dry them in a 40℃ oven for 24h. The dried ginseng fragments are collected in a 2ml centrifuge tube, added with zirconium oxide beads, and ground into powder by a tissue crusher (60HZ, 2min) for later saponin content detection (saponin component extraction and content detection are the same as above).

[0070] (2) Results of G198 bacteria promoting the accumulation of saponin content in potted ginseng

[0071] The G198 strain significantly promoted the increase of the total content of saponins. The total content of saponins in the bacterial treatment group was significantly higher than that in the control group, with a significant difference (P≤0.05), and the total content increased by 91.2% (Table 5-1 and Table 5-2). From the analysis of the content of single saponins, the contents of Rg1, Re, Rc, Ro, Rb1, Rd, Rh1, R1, F2 and 20DRf were significantly increased (P≤0.05).

[0072] Table 5. Results of G198 strain promoting the accumulation of ginsenoside content in potted plants

[0073]

[0074]

[0075] Note: Different lowercase letters represent significant differences at the 0.05 level.

Claims

1. A strain of Bradyrhizobium mesogenum ( Mesorhizobium sp. ) G198, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No: 30089, and the deposit date is March 21, 2024.

2. The mesorhizobium according to claim 1 ( Mesorhizobium sp. ) Bacterial agent prepared by G198.

3. The method for preparing the bacterial agent according to claim 2, characterized in that: The method comprises inoculating the mesorhizobium G198 with a preservation number of CGMCC No: 30089 into a 1 / 2 TSB liquid medium and culturing the culture medium until the strain concentration is 1*10 8 CFU / mL, the bacterial solution is centrifuged, repeatedly eluted with sterile distilled water, and centrifuged to obtain clean bacteria without culture medium components, and finally sterile distilled water is added to resuspend to obtain the bacterial agent; wherein the culture conditions are: temperature 25-30 ℃, rotation speed 100-150 rpm, dark culture 70-75 h.

4. The preparation method according to claim 3, characterized in that: The culture conditions are: temperature 28°C, rotation speed 120 rpm, and dark culture for 72 h.

5. The mesorhizobium according to claim 1 ( Mesorhizobium sp. ) G198 or the use of the bacterial agent described in claim 2 in promoting the accumulation of ginseng saponin components or in preparing products that promote the accumulation of ginseng saponin components; the ginsenoside components are selected from one or more of Rg1, Re, Rc, Ro, Rb1, Rd, Rh1, R1, F2, Rh2, Rh4 and CK.

6. The use according to claim 5, characterized in that The mesorhizobium of claim 1 ( Mesorhizobium sp. ) Use of the bacterial agent described in G198 or claim 2 in increasing the content of Rg1, Re, Rc, Ro, Rb1, Rd, Rh1, R1 and F2 in potted ginseng.

7. The mesorhizobium of claim 1 ( Mesorhizobium sp. ) Use of G198 or the bacterial agent described in claim 2 to increase the content of saponins Rh1, Rh2, Rh4, F2 and CK in ginseng adventitious roots.

8. A method for increasing the content of saponins in ginseng adventitious roots, characterized in that: comprising the mesorhizobium ( Mesorhizobium sp. )G198 was added at a final concentration of 1*10 7 CFU / mL is inoculated into the ginseng adventitious root liquid culture system, and co-cultured for 10 days or more; the ginseng adventitious root culture liquid system is prepared by the following method: 30 mL of adventitious root culture medium is added to a 50 mL culture bottle, 1 g of ginseng adventitious roots are inoculated, and liquid culture is carried out for 18-22 days or cultured until the ginseng adventitious root biomass reaches 2.5-3.5 g fresh weight; the adventitious root culture medium formula is as follows: 30 g sucrose, 5 mg IBA hormone, 3.35 g MS powder, 1 L double distilled water, pH = 6.0; the liquid culture conditions are 25°C constant temperature dark culture, and a rotation speed of 120 rpm; the co-culture conditions are a temperature of 25-30°C, a rotation speed of 100-150 rpm, and dark culture; wherein the ginseng adventitious root saponin components are selected from Rh1, Rh2, Rh4, F2 and CK.

9. A method for increasing the content of ginsenosides in potted plants, characterized in that: The method comprises applying the mesorhizobium ( Mesorhizobium sp. ) G198 clean bacterial solution, 50 mL of bacterial solution per pot each time, to ensure that the concentration of the inoculated soil bacterial solution is 1*10 7 CFU / g soil; wherein the potted ginsenoside components are selected from Rg1, Re, Rc, Ro, Rb1, Rd, Rh1, R1 and F2.

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