Ginsenoside-producing variant flavobacterium dg-16 and application thereof in alleviating continuous cropping obstacles of angelica sinensis and improving quality of angelica sinensis

Abstract

The application belongs to the technical field of biological bacterial agents, and particularly relates to a ginsenoside variant flavobacterium DG-16 and application of the ginsenoside variant flavobacterium DG-16 in relieving continuous cropping obstacles of angelica and improving quality of the angelica. The ginsenoside variant flavobacterium DG-16 can promote growth of the angelica by increasing contents of jasmonic acid and cell division hormone, reducing contents of gibberellin and salicylic acid hormone, and promoting circulation and utilization of nitrogen. In addition, the ginsenoside variant flavobacterium DG-16 can significantly increase chlorophyll content, which is beneficial to photosynthesis of the angelica, effectively changes physiological and biochemical states of the angelica, and can resist root rot of the angelica, so as to relieve the continuous cropping obstacles and improve the quality, and the effect is significant.

Description

Technical Field

[0001] This invention belongs to the field of biological agent technology, specifically relating to a strain of ginsenoside mutant Flavobacterium DG-16 and its application in alleviating the obstacles of continuous cropping of Angelica sinensis and improving the quality of Angelica sinensis. Background Technology

[0002] Angelica sinensis is a plant belonging to the Apiaceae family. Angelica sinensis(Oliv. ) The dried root of Angelica sinensis has the effects of nourishing blood, promoting blood circulation, regulating menstruation and relieving pain, and moistening the intestines and relieving constipation. In China, Angelica sinensis is mainly produced in Gansu, such as Minxian and Dangchang in Gansu, which are the authentic producing areas of Angelica sinensis. However, in recent years, due to continuous cropping obstacles, diseases such as root rot have occurred frequently, with the incidence rate in some areas exceeding 50%, which has seriously affected the development of the industry.

[0003] Currently, the main methods used to improve soil physical and chemical properties and reduce pests and diseases include crop rotation, increased application of organic fertilizers, soil disinfectants and fungicides, and soil testing-based fertilization. While these methods have achieved some results, the long rotation cycle affects short-term economic benefits and makes them difficult for producers to accept. Furthermore, chemical disinfectants may cause environmental pollution, and long-term use can lead to drug resistance in pathogens. Microbial control, with its green, environmentally friendly, and highly efficient advantages, is currently a hot research topic.

[0004] Ginsenoside variant Flavobacterium ( Flavobacterium ginsenosidimutans ) belongs to the genus Flavobacterium ( Flavobacterium Bacteria. Flavobacterium bacteria have plant growth-promoting and disease-controlling effects. For example, in some studies known to the inventors, a strain of Flavobacterium has been reported. Flavobacterium F-55 exhibits inhibitory activity against pathogenic strains of *Pseudomonas syringae* in kiwifruit and promotes plant growth; it can be used as a biocontrol agent to prevent bacterial canker in kiwifruit. In other studies known to the inventors, a strain of *Flavobacterium tumefaciens* was reported. Flavobacterium erigeronis The strain numbered 3-218 possesses the functions of producing iron carriers, promoting growth, and alleviating salt-alkali stress in plants. It can be used as a microbial organic fertilizer to improve soil fertility, alleviate crop salt-alkali stress, and enhance crop adaptability to salt-alkali environments. However, there are currently no reports on Flavobacterium bacteria that can alleviate continuous cropping obstacles and improve the quality of Angelica sinensis. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of ginsenoside mutant Flavobacterium DG-16 and its application in alleviating the obstacles of continuous cropping of Angelica sinensis and improving the quality of Angelica sinensis. The ginsenoside mutant Flavobacterium DG-16 and the bacterial agent containing the ginsenoside mutant Flavobacterium DG-16 can alleviate the obstacles of continuous cropping of Angelica sinensis and improve the quality of Angelica sinensis, with significant effects.

[0006] This invention provides a strain of ginsenoside mutant Flavobacterium DG-16, the preservation number of which is CGMCC No.29478.

[0007] The present invention also provides a bacterial agent containing the ginsenoside mutant Flavobacterium DG-16 described in the above technical solution.

[0008] Preferably, the effective viable bacterial concentration of ginsenoside-mutant Flavobacterium DG-16 in the bacterial agent is 2 × (10⁻⁶)⁻¹. 8 ~10 9 CFU / mL.

[0009] Preferably, the active ingredient of the microbial agent includes ginsenoside mutant Flavobacterium DG-16, fermentation broth of ginsenoside mutant Flavobacterium DG-16, or suspension of ginsenoside mutant Flavobacterium DG-16.

[0010] The present invention also provides the application of the ginsenoside mutant Flavobacterium DG-16 or the bacterial agent described in the above technical solution in at least one of improving the quality of Angelica sinensis, alleviating the obstacle of continuous cropping of Angelica sinensis, promoting the growth of Angelica sinensis and resisting root rot.

[0011] Preferably, improving the quality of Angelica sinensis includes increasing the content of its effective components.

[0012] Preferably, the promotion of Angelica sinensis growth includes at least one of the following: 1) Increase the content of endogenous jasmonic acid and / or cytokinin-like hormones in Angelica sinensis; 2) Reduce the content of angelica gibberellin and / or salicylate hormones; 3) Promote nitrogen cycling and utilization; 4) Increase the chlorophyll content of Angelica sinensis; 5) Increase at least one of the following: weight of 100 Angelica plants, diameter of rhizome, and length of stem.

[0013] The present invention also provides a method for improving the quality of Angelica sinensis, alleviating continuous cropping obstacles of Angelica sinensis, promoting the growth of Angelica sinensis, and resisting root rot, comprising the following steps: The above-ground parts of Angelica sinensis were sprayed with a bacterial agent containing ginsenoside mutant Flavobacterium DG-16, the preservation number of which is CGMCC No.29478.

[0014] Preferably, based on the viable count of the ginsenoside mutant Flavobacterium DG-16, the spraying dosage is (1.5~2.5) × (10⁻⁶)⁻¹. 11 ~10 12 CFU / mu; the spraying period is 1 to 4 months after the transplanting of Angelica sinensis seedlings, spraying once every 4 weeks, for a total of 3 to 4 sprays.

[0015] Preferably, the spraying method includes foliar spraying.

[0016] Beneficial effects: This invention provides a strain of ginsenoside mutant Flavobacterium DG-16 and its application in alleviating continuous cropping obstacles and improving the quality of Angelica sinensis. The present invention isolates a strain of ginsenoside mutant Flavobacterium DG-16, names it DG-16, and preserves it biologically. The ginsenoside mutant Flavobacterium DG-16 promotes the growth of Angelica sinensis by increasing the content of jasmonic acid and cytokinin-like hormones, decreasing the content of gibberellin and salicylic acid-like hormones, and promoting nitrogen cycling and utilization. In addition, the ginsenoside mutant Flavobacterium DG-16 can significantly increase chlorophyll content, which is beneficial for photosynthesis in Angelica sinensis, effectively changing the physiological and biochemical state of Angelica sinensis, and simultaneously resisting root rot disease, thus alleviating continuous cropping obstacles and improving quality, with significant effects.

[0017] Biological Preservation Information Ginsenoside variant Flavobacterium DG-16, biologically classified as Flavobacterium ginsenosidimutans It was deposited on January 2, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCCNo. 29478. Detailed Implementation

[0018] This invention provides a strain of ginsenoside mutant Flavobacterium DG-16, the preservation number of which is CGMCC No.29478.

[0019] In this invention, the *Flavobacterium ginsenoside mutant* DG-16 was isolated from medicinal plants grown in Minxian County, Dingxi City, Gansu Province, China, a high-altitude and cold region. It was identified as *Flavobacterium ginsenoside mutant* through colony characteristics, physicochemical properties, and 16S rDNA sequence. The colony characteristics of *Flavobacterium ginsenoside mutant* DG-16 on LB solid medium are: yellowish-brown, translucent, smooth and moist surface, and irregular edges. The 16S rDNA sequence of *Flavobacterium ginsenoside mutant* DG-16 is shown in SEQ ID NO:1.

[0020] The present invention also provides a bacterial agent containing the ginsenoside mutant Flavobacterium DG-16 described in the above technical solution.

[0021] As one implementation method, the effective viable bacterial concentration of ginsenoside-mutant Flavobacterium DG-16 in the bacterial agent is 2 × (10⁻⁶)⁻¹. 8 ~10 9As one embodiment, the effective component of the bacterial agent can be ginsenoside mutant Flavobacterium DG-16, the fermentation broth of ginsenoside mutant Flavobacterium DG-16, or a suspension of ginsenoside mutant Flavobacterium DG-16.

[0022] In one embodiment, the preparation method of the fermentation broth of *Flavobacterium ginsenoside mutant* DG-16 according to the present invention includes the following steps: inoculating *Flavobacterium ginsenoside mutant* DG-16 into a liquid fermentation medium for fermentation culture to obtain the fermentation broth of *Flavobacterium ginsenoside mutant* DG-16. In one embodiment, the liquid fermentation medium of the present invention can be PDB medium. In one embodiment, the fermentation culture temperature can be 25~30℃ or 28℃. In one embodiment, the fermentation culture time can be 1~3 days or 2 days. In one embodiment, the shaking frequency of the fermentation culture can be 150~200 r / min; in another embodiment, the shaking frequency of the fermentation culture can be 180 r / min.

[0023] The present invention also provides the application of the ginsenoside mutant Flavobacterium DG-16 or the bacterial agent described in the above technical solution in at least one of improving the quality of Angelica sinensis, alleviating the obstacle of continuous cropping of Angelica sinensis, promoting the growth of Angelica sinensis and resisting root rot.

[0024] In one embodiment, improving the quality of Angelica sinensis includes increasing the content of its effective components; in another embodiment, improving the quality of Angelica sinensis includes increasing the content of one or more of the following components: ferulic acid, ligustilide H, ferulic acid coniferyl ester, and ligustilide.

[0025] As one implementation method, the method for promoting the growth of Angelica sinensis according to the present invention includes at least one of the following: 1) Increase the content of endogenous jasmonic acid and / or cytokinin-like hormones in Angelica sinensis; 2) Decrease the content of gibberellin and / or salicylic acid-like hormones in Angelica sinensis; 3) Promote nitrogen cycling and utilization; 4) Increase the chlorophyll content of Angelica sinensis; 5) Increase at least one of the following: weight per 100 plants, rhizome diameter, and stem length. As one embodiment, increasing the content of endogenous jasmonic acid and / or cytokinin-like hormones in Angelica sinensis includes increasing the content of one or more hormones selected from 6-furfurylaminopurine, 2-methylthioisopentenyladenine nucleoside, dihydrojasmonic acid, and 12-hydroxyjasmonic acid. As one embodiment, decreasing the content of gibberellin and / or salicylic acid-like hormones in Angelica sinensis includes decreasing the content of one or more hormones selected from gibberellin-4, cinnamic acid, and salicylic acid-2-O-β-glucoside.

[0026] The present invention also provides a method for improving the quality of Angelica sinensis, alleviating continuous cropping obstacles of Angelica sinensis, promoting the growth of Angelica sinensis, and resisting root rot, comprising the following steps: The above-ground parts of Angelica sinensis were sprayed with a bacterial agent containing ginsenoside mutant Flavobacterium DG-16, the preservation number of which is CGMCC No.29478.

[0027] As one implementation method, based on the viable count of the ginsenoside mutant Flavobacterium DG-16, the spraying dosage is (1.5~2.5) × (10^6)^2. 11 ~10 12 The concentration of the microbial agent is 5 × (10⁻⁶ CFU / mu; the spraying period is 1-4 months after transplanting the Angelica sinensis seedlings, sprayed once every 4 weeks, for a total of 3-4 sprays. In the embodiments of the present invention, the concentration of the microbial agent during spraying is 5 × (10⁻⁶ CFU / mu). 6 ~10 7 The concentration of the microbial agent is CFU / mL; the spraying dosage is 35-45 L / mu, or 40 L / mu. In one embodiment, the spraying period is 1-4 months after transplanting the Angelica sinensis seedlings, spraying once every 4 weeks for a total of 3-4 times; in another embodiment, the spraying frequency is 4 times. In one embodiment, the spraying method includes foliar spraying. In one embodiment, the microbial agent of this invention can be the fermentation broth of *Flavobacterium ginsenoside* DG-16, the characteristics of which have been defined in the above technical solutions and will not be repeated here.

[0028] The ginsenoside mutant Flavobacterium DG-16 microbial agent of this invention possesses nitrogen-fixing, heparin-producing, and nitrifying properties, as well as high activity of ACC deaminase, cellulase, laccase, and catalase. Simultaneously, this microbial agent can increase the content of jasmonic acid and cytokinin-like hormones while decreasing the content of gibberellins and salicylic acid-like hormones; promote nitrogen cycling and utilization, significantly increase chlorophyll content, which is beneficial for photosynthesis in Angelica sinensis; effectively alter the physiological and biochemical state of Angelica sinensis, promote its growth, and alleviate continuous cropping obstacles while improving quality.

[0029] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] The Angelica sinensis seedlings used in the following examples were one-year-old Angelica sinensis seedlings of the Minxian Traditional Chinese Medicine Production Technology Guidance Station. The experiment was conducted in 2024 in Shili Town, Minxian County, Gansu Province. The soil at the transplanting site was dark brown soil with a pH of 6.8. The base fertilizer consisted of 1 ton / mu of organic fertilizer (organic matter ≥40%, N+P2O5+K2O ≥4%) and 20 kg / mu of balanced compound fertilizer (17-17-17).

[0031] All reagents and culture media used in the experiment were chemically pure. The activity assay kits for cellulase, laccase, xylanase, peroxidase, ACC deaminase, glutamine synthase, glutamate synthase, nitrite reductase, nitrate reductase, and phenylalanine ammonia-lyase, as well as the assay kits for ABTS, DPPH, ferrophosphate, malondialdehyde, ammonium nitrogen, nitrate nitrogen, amino nitrogen, soluble sugars, chlorophyll, flavonoids, nitrite nitrogen, and nitrate nitrogen were purchased from Beijing Box Biotechnology Co., Ltd.

[0032] The composition of the culture medium used in the following examples: PDB medium: 200 g / L potato, 20 g / L glucose, natural pH.

[0033] LB medium: tryptic peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH adjusted to 7.0 ± 0.2.

[0034] Modified Stephenson medium: ammonium sulfate 2 g / L, manganese sulfate 0.01 g / L, sodium dihydrogen phosphate 0.25 g / L, magnesium sulfate 0.03 g / L, calcium carbonate 0.5 g / L, dipotassium hydrogen phosphate 0.75 g / L, pH adjusted to 8.2.

[0035] NBRIP solid medium: glucose 10 g / L, calcium phosphate 5 g / L, magnesium chloride 5 g / L, magnesium sulfate heptahydrate 0.25 g / L, potassium chloride 0.2 g / L, ammonium sulfate 0.1 g / L, agar 15 g / L, pH 7.0 ± 0.2.

[0036] Simon's citrate agar medium: sodium chloride 5.0 g / L, magnesium sulfate 0.2 g / L, ammonium dihydrogen phosphate 1.0 g / L, dipotassium hydrogen phosphate 1.0 g / L, sodium citrate 5.0 g / L, agar 20 g / L, 0.2% bromothymol blue solution 40 mL / L, adjust pH to 6.8 ± 0.2.

[0037] Glucose-peptone aqueous medium: glucose 0.5 g / L, peptone 0.5 g / L, dipotassium hydrogen phosphate 0.2 g / L, pH adjusted to 7.2~7.4.

[0038] Peptone amination medium: 5 g / L peptone, 0.5 g / L dipotassium hydrogen phosphate, 0.25 g / L sodium chloride, 0.5 g / L magnesium sulfate heptahydrate, 0.01 g / L ferrous sulfate, adjusted to pH 7.2.

[0039] Example 1 The isolation and identification of ginsenoside mutant Flavobacterium DG-16 were performed using the following steps: A strain of fungus was isolated from Angelica sinensis, a medicinal plant growing in Minxian County, Dingxi City, Gansu Province, China, by members of the inventor's research team.

[0040] The colony characteristics of the strain are as follows: The isolated strain was inoculated into LB solid medium and cultured at 30°C for 2 days. The colony morphology of the strain was: yellowish-brown, translucent, smooth and moist surface, and irregular edges.

[0041]

[0042] Example 2 The functional assay of ginsenoside mutant Flavobacterium DG-16 was performed using the following steps: 1) Determination of phosphorus solubilization characteristics: An appropriate amount of *Flavobacterium ginsenoside mutant* DG-16 strain was inoculated into LB medium and cultured at 28℃ and 180 r / min for 2 days. 10 μL of *Flavobacterium ginsenoside mutant* DG-16 bacterial culture was then inoculated onto NBRIP solid medium, and this inoculation was repeated three times. The medium was then incubated at 30℃. The presence and size of phosphorus-solubilizing zones on the medium were observed periodically over 7 days. The phosphorus solubilization capacity of the strain was determined based on the size of the phosphorus-solubilizing zones.

[0043] The results showed that the ginsenoside mutant Flavobacterium DG-16 had no phosphorus solubilizing ability.

[0044] 2) Nitrogen fixation characteristics determination: An appropriate amount of ginsenoside mutant Flavobacterium DG-16 strain was inoculated into LB medium and cultured at 30℃ and 180r / min for 2 days with shaking. An equal amount of 10μL of bacterial solution was inoculated into sterile Ashby medium with 0.2% Congo red chromogenic agent. The inoculation was repeated three times and cultured at 30℃ for 3-5 days. The growth of colonies and color changes on the medium were observed for qualitative detection.

[0045] The results showed that the ginsenoside mutant Flavobacterium DG-16 strain could grow and had a clear zone, indicating nitrogen fixation ability.

[0046] 3) Citrate utilization: Take an appropriate amount of ginsenoside mutant Flavobacterium DG-16 and inoculate it into the center of Simon's citrate agar medium using an inoculation loop. Repeat the inoculation three times to ensure even colony distribution. Invert the inoculated plate in a 30℃ incubator and incubate for 24-48 hours. Observe whether a color change occurs around the colonies on the plate; turning blue indicates a positive result.

[0047] The results showed that ginsenoside mutant Flavobacterium DG-16 can utilize citrate.

[0048] 4) Nitrification power determination: Ginsenoside mutant Flavobacterium DG-16 was inoculated into LB medium and cultured at 30℃ and 180 r / min for 2 days with shaking. The bacterial cells were collected by centrifugation and resuspended in sterile water to OD. 600 The concentration was set to 1.0, and then inoculated with sterilized modified Stephenson's medium. The mixture was incubated at 30°C with shaking at 180 rpm for 2 days, and the OD was measured. 600 The nitrifying power was determined according to the methods described in the nitrite nitrogen and nitrate nitrogen kits. The result was then divided by the turbidity of the corresponding bacterial suspension and standardized to the nitrifying power per unit turbidity. Each strain was repeated three times, and the average value was calculated.

[0049] The results showed that the nitrification power of ginsenoside mutant Flavobacterium DG-16 was 1.358±0.09%.

[0050] 5) Determination of relative content of ferrophilic acid: Ginsenoside mutant Flavobacterium DG-16 was inoculated into LB medium and centrifuged to obtain the ferrophilic acid fermentation supernatant (SCS). SCS and cyanobacterium sulfate S (CAS) solutions were mixed at a volume ratio of 1:1, and the mixture was incubated at 37°C in the dark for 0.5 h before the OD of the mixture was measured. 630 The relative heptaphile content of the strain was calculated using the following formula: Relative heptaphile content of the strain = (Ar - As) / Ar × 100%, where Ar is the OD of the reference compound. 630 (A mixture of blank control and CAS); As is the sample OD 630 (A mixture of strains SCS and CAS). The calculated result is divided by the turbidity of the corresponding bacterial solution and standardized to the relative content of ferrophile in the bacterial solution per unit turbidity.

[0051] The results showed that the relative content of ginsenoside mutant Flavobacterium DG-16 was 45.4%.

[0052] 6) Determination of ACC deaminase activity of the strain: Ginsenoside mutant Flavobacterium DG-16 was cultured in LB medium at 30℃ and 180r / min for 1 day with shaking. Then, it was centrifuged at 4℃ and 8000r / min for 10 min, the supernatant was discarded, and the bacterial cells were washed twice with ADF medium without (NH4)2SO4. After centrifugation, the bacterial cells were resuspended in ADF medium and cultured at 30℃ and 180r / min for 1 day. After centrifugation at 8000 rpm for 10 min at 4 °C, the supernatant was discarded and the bacterial cells were collected. The cells were washed twice with 0.1 mol / L Tris-HCl buffer (pH 7.6), centrifuged again, and resuspended in 0.2 mL of Tris-HCl buffer (pH 8.5). A small amount of toluene was added, and the cells were sonicated to disrupt the cell structure. 20 μL of 0.5 mol / L ACC was added, and the mixture was incubated at 30 °C for 15 min. Then, 0.3 mL of dinitrophenylhydrazine was added, the container was capped, and the mixture was incubated at 30 °C for 0.5 h. The reaction was then terminated by adding 2 mL of 2 mol / L NaOH, and the absorbance at 540 nm was measured. The ACC deaminase activity was calculated as the amount of α-butanone produced per minute (μmol / min). The calculated result was divided by the turbidity of the corresponding bacterial suspension and standardized to the ACC deaminase activity per unit turbidity of the bacterial suspension.

[0053] The results showed that the deaminase activity of ginsenoside mutant Flavobacterium DG-16 was 0.445 μmol / min.

[0054] 7) Determination of enzyme activity of the strain: *Flavobacterium ginsenoside mutant* DG-16 was inoculated into 50 mL of sterile LB medium and cultured at 30℃ with shaking at 180 r / min for 2 days. The cells were then filtered, collected, and resuspended in sterile water to a turbidity OD value. 600 The bacterial culture to be tested was obtained by setting the bacterial culture to enzyme extract at a volume ratio of 1:10. Cells were disrupted by sonication in an ice bath, followed by centrifugation at 8000 rpm for 10 min at 4°C. The supernatant was placed on ice for testing. The activity of peroxidase, cellulase, neutral xylanase, and laccase was determined using spectrophotometry according to the instructions of the kits, with three replicates. The results were divided by the turbidity of the corresponding bacterial culture and standardized to the enzyme activity per unit turbidity.

[0055] The results showed that the peroxidase activity of *Flavobacterium ginsenoside* DG-16 was 1.048±0.042 U / mL, the neutral xylanase activity was 0 U / mL, the cellulase activity was 4.810±0.0114 U / mL, and the laccase activity was 19.558±0.178 U / mL.

[0056] 8) Methyl red test: Use a sterile inoculation loop to inoculate ginsenoside mutant Flavobacterium DG-16 into sterile glucose peptone water medium, with no medium inoculated as a control. Incubate at 30℃ for 2-5 days, add 5 drops of methyl red reagent to the medium and observe the color change. A red color indicates a negative result.

[0057] The results showed that ginsenoside mutant Flavobacterium DG-16 could not break down glucose to produce acid.

[0058] 9) Ammoniation test: Inoculate the fermentation broth of *Flavobacterium ginsenoside mutant* DG-16 into peptone ammoniation medium, and inoculate 6 dilutions (10⁻⁶). -2 10 -3 10 -4 10 -5 10 -6 10 -7 Three tubes were inoculated for each dilution, and one tube of culture medium was inoculated with sterile water as a control. The cultures were incubated at 30°C. The turbidity of the culture medium was checked on days 3 and 5. On day 7, five drops of culture medium were placed on a white porcelain colorimetric plate, two drops of Nessler's reagent were added, and the presence of a brownish color was checked to determine if ammonia had been produced.

[0059] The results showed that the ginsenoside mutant Flavobacterium DG-16 had a strong ammonification ability.

[0060] 10) Catalase test: Pick a colony of *Bacillus ginsenoside variant* DG-16, place it on a clean glass slide, and add an appropriate amount of 3% hydrogen peroxide solution. A positive result is indicated by the generation of a large number of bubbles within 1 minute.

[0061] The results showed that the catalase (catalase) activity of ginsenoside mutant Flavobacterium DG-16 was relatively strong.

[0062] 11) Determination of antagonistic characteristics of strains: The antagonistic activity between ginsenoside variant Flavobacterium DG-16 and pathogenic fungus Fusarium solani was determined by plate confrontation culture method: the pathogen was inoculated in the center of PDA medium, and the strain was inoculated in equal amounts at symmetrical distances above, below or on both sides. The medium was incubated at 30℃ for 5-7 days, and the colony growth and inhibition zone size were observed. The inhibition rate was calculated according to the following formula: inhibition rate = (inhibition zone radius - antagonistic bacteria radius) / antagonistic bacteria radius × 100%.

[0063] The results showed that ginsenoside mutant Flavobacterium DG-16 could inhibit the growth of Fusarium rotundum, and the inhibition rate was 12.47±0.01%.

[0064] Example 3 The effects of ginsenoside mutant Flavobacterium DG-16 on alleviating continuous cropping obstacles and improving quality are investigated through the following steps: The experimental group is set up as follows: Treatment group (T): The fermentation broth of *Flavobacterium ginsenoside mutant* DG-16 cultured in PDB medium at 28℃ and 180r / min for 2 days was diluted 40 times with sterile water to achieve an effective viable bacterial count of 5 × 10⁻⁶. 6 ~10 7 CFU / mL. Apply 40L per acre.

[0065] Control group (CK): PDB medium without bacteria was diluted by the same factor as the treatment group and sprayed at 40L per acre.

[0066] The experiment employed a single-factor completely randomized design, continuing angelica cultivation on plots where a previous crop of commercial angelica had been harvested. Two treatments, T and CK, were established, with three replicates per treatment. Each plot was 30 m² in size. 2 (4m×7.5m). Angelica seedlings were transplanted in early April 2024. Foliar spraying was applied 4 times from May to August, once every 4 weeks. The volume of spraying was the same for different plots.

[0067] Field management was carried out according to standard procedures.

[0068] In late August, 5 days after the fourth treatment, 100 Angelica plants were randomly selected from each plot, leaves were picked, and rhizosphere soil corresponding to the Angelica plants was collected, mixed separately, and brought back to the laboratory for later use.

[0069] By the end of October 2024, the weight of 100 plants, root rot and other growth indicators were statistically analyzed and measured, and the content of effective components of Angelica sinensis was determined by high performance liquid chromatography.

[0070] Table 1 shows the weight per 100 plants and growth indicators of Angelica sinensis after treatment with ginsenoside mutant Flavobacterium DG-16, and Table 2 shows the effective components of Angelica sinensis.

[0071] Table 1 Effects of microbial inoculants on growth and disease-related indicators of Angelica sinensis (x±se, n=3)

[0072] Table 2 Effects of microbial inoculants on the effective components of Angelica sinensis (x±se, n=3)

[0073] Note: Student's T-test was performed on the data; different lowercase letters indicate significant differences. P <0.05), the same applies below.

[0074] Table 1 shows that compared with the control group CK, the T group treatment described in this invention significantly increased the weight of 100 Angelica sinensis plants ( ). P <0.05), indicating that the treatment group T described in this invention effectively alleviated the continuous cropping obstacle of Angelica sinensis and promoted its growth. Table 2 shows that compared with the control group CK, the content of ferulic acid, ligustilide H, ferulic acid coniferyl ester, and ligustilide in the treatment group T of this invention was significantly increased, indicating that the treatment group T described in this invention effectively improved the quality of Angelica sinensis.

[0075] Example 4 The effects of ginsenoside mutant Flavobacterium DG-16 on endogenous hormone levels in Angelica sinensis leaves were investigated using the following steps: The test setup and test plan are the same as in Example 3. (1) Detection of endogenous hormones: The content of endogenous hormones in Angelica sinensis leaves was determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS) (see Table 3). Leaves from different samples were sent to a third party for detection. The indicators were: 109 substances in total, including 9 major categories of plant hormones and intermediate metabolites, namely auxin, cytokinins (CKs), abscisic acid (ABA), jasmonates (JAs), salicylic acid (SA), gibberellins (GAs), 1-aminocyclopropanecarboxylic acid (ACC), strigolactones (SLs), and melatonin (MLT). The most important substances that effectively distinguished the differences between the T and CK groups were screened by Log2FC value (Log2FC>1). The results of the differential metabolite screening are shown in Table 3.

[0076] Table 3. Endogenous hormones in Angelica sinensis leaves

[0077] Note: Log2FC: The difference factor is the logarithm to base 2, the same applies below. Inf represents infinity, -Inf represents infinitesimal. Table 3 shows that the hormone content in the treatment group T described in this invention differed significantly from that in the control group (CK). A total of 10 metabolic species showed significant differences, with 5 significantly upregulated and 5 significantly downregulated. Treatment with *Flavobacterium ginsenoside* DG-16 increased the content of N-(3-indoleacetyl)-L-phenylalanine, 6-furfurylaminopurine, 2-methylthioisopentenyladenine nucleoside, dihydrojasmonic acid, and 12-hydroxyjasmonic acid, while decreasing the content of indole-3-acrylic acid, gibberellin 4, cinnamic acid, salicylic acid-2-O-β-glucoside, and 5-deoxystreptococcal alcohol. This indicates that the *Flavobacterium ginsenoside* inoculum can activate *Angelica sinensis* growth-promoting hormones and disease-resistant hormones, inhibit salicylic acid hormones and gibberellins, thereby promoting growth, improving disease resistance, alleviating continuous cropping obstacles, and improving quality.

[0078] (2) Determination of physiological and biochemical indicators of leaves: The equivalent values ​​of ABTS scavenging capacity, DPPH scavenging capacity, total phenols, malondialdehyde, glutamine synthase, glutamate synthase, nitrite reductase, nitrate reductase, ammonium nitrogen, nitrate nitrogen, amino nitrogen, soluble sugar, chlorophyll, phenylalanine ammonia-lyase (PAL) activity, and flavonoids in Angelica sinensis leaves were determined by spectrophotometry according to the kit instructions. Each treatment was repeated 3 times, and the results with significant differences are shown in Table 4.

[0079] Table 4. Detection of physiological and biochemical indicators of leaves (x±se, n=3)

[0080] As shown in Table 3, there were significant differences in the relevant physiological and biochemical indicators of Angelica sinensis leaves. The contents of glutamate synthase, nitrate reductase, nitrate nitrogen, amino nitrogen, and chlorophyll increased significantly, indicating that the nitrogen assimilation efficiency and photosynthetic pigment synthesis were improved after the T group treatment described in this invention, thus promoting the growth of Angelica sinensis.

[0081] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A ginsenoside mutant Flavobacterium that alleviates continuous cropping obstacles and improves the quality of Angelica sinensis ( Flavobacterium ginsenosidimutans The ginsenoside mutant Flavobacterium DG-16 has the accession number CGMCC No. 29478.

2. A bacterial agent containing the ginsenoside mutant Flavobacterium DG-16 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The effective viable bacterial concentration of *Flavobacterium ginsenoside mutant* DG-16 in the bacterial agent is 2 × (10⁻⁶). 8 ~10 9 CFU / mL.

4. The microbial agent according to claim 2, characterized in that, The active ingredients of the microbial agent include ginsenoside mutant Flavobacterium DG-16, fermentation broth of ginsenoside mutant Flavobacterium DG-16, or suspension of ginsenoside mutant Flavobacterium DG-16.

5. A method, wherein the method comprises at least one of improving the quality of Angelica sinensis, alleviating the continuous cropping obstacles of Angelica sinensis, promoting the growth of Angelica sinensis, and resisting root rot, characterized in that, Includes the following steps: The above-ground parts of Angelica sinensis were sprayed with a bacterial agent containing ginsenoside mutant Flavobacterium DG-16, the preservation number of which is CGMCC No.29478.

6. The method according to claim 5, characterized in that, Based on the viable count of the aforementioned ginsenoside mutant Flavobacterium DG-16, the spraying dosage is 1.5 × 10⁻⁶. 11 ~2.5×10 12 CFU / mu; the spraying period is 1 to 4 months after the transplanting of Angelica sinensis seedlings, spraying once every 4 weeks, for a total of 3 to 4 sprays.

7. The method according to claim 5 or 6, characterized in that, The spraying method includes foliar spraying.

8. The method according to claim 5, characterized in that, Improving the quality of Angelica sinensis includes increasing the content of its effective components.

9. The method according to claim 5, characterized in that, The promotion of Angelica sinensis growth includes at least one of the following: 1) Increase the content of endogenous jasmonic acid and / or cytokinin-like hormones in Angelica sinensis; 2) Reduce the content of angelica gibberellin and / or salicylate hormones; 3) Promote nitrogen cycling and utilization; 4) Increase the chlorophyll content of Angelica sinensis; 5) Increase the weight of 100 Angelica plants and / or the thickness of the rhizome.

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