Flavobacterium ginsenoside variant DG-16 and application thereof in relieving continuous cropping obstacle of angelica sinensis and improving quality of angelica sinensis

Abstract

The invention belongs to the technical field of biological agents, and particularly relates to ginsenoside variant flavobacterium DG-16 and application thereof in relieving continuous cropping obstacles of angelica sinensis and improving the quality of the angelica sinensis. According to the ginsenoside variant flavobacterium DG-16 disclosed by the invention, the growth of angelica sinensis is promoted by increasing the contents of jasmonic acid and cytokinin hormones, reducing the contents of gibberellin and salicylic acid hormones and promoting the circulation and utilization of nitrogen; in addition, the flavobacterium ginsenoside variant DG-16 can significantly increase the chlorophyll content, is beneficial to photosynthesis of the angelica sinensis, effectively changes the physiological and biochemical states of the angelica sinensis, can resist root rot of the angelica sinensis so as to relieve successive cropping obstacles and improve the quality, and has a significant effect.

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 Flavobacterium 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] The application provides a ginsenoside variant flavobacterium DG-16, and the preservation number of the ginsenoside variant flavobacterium DG-16 is CGMCC No.29478.

[0007] The application further provides a microbial agent containing the ginsenoside variant flavobacterium DG-16.

[0008] Preferably, the effective viable cell concentration of the ginsenoside variant flavobacterium DG-16 in the microbial agent is 2×(10 8 ~10 9 ) CFU / mL.

[0009] Preferably, the effective components of the microbial agent include the ginsenoside variant flavobacterium DG-16, a fermentation liquor of the ginsenoside variant flavobacterium DG-16 or a suspension of the ginsenoside variant flavobacterium DG-16.

[0010] The application further provides application of the ginsenoside variant flavobacterium DG-16 or the microbial agent in at least one of improving the quality of angelica, relieving the continuous cropping obstacles of angelica, promoting the growth of angelica and resisting root rot of angelica.

[0011] Preferably, the improvement of the quality of angelica includes improvement of the content of effective components of angelica.

[0012] Preferably, the promotion of the growth of angelica includes at least one of the following: 1) improvement of the content of endogenous jasmonic acid and / or cytokinin of angelica; 2) reduction of the content of gibberellin and / or salicylic acid of angelica; 3) promotion of the circulation and utilization of nitrogen; 4) improvement of the content of chlorophyll of angelica; 5) improvement of at least one of the following: the weight of 100 plants, the length of rhizome and the length of stem of angelica.

[0013] The application further provides a method for at least one of improving the quality of angelica, relieving the continuous cropping obstacles of angelica, promoting the growth of angelica and resisting root rot of angelica, and the method comprises the following steps: Spraying a microbial agent containing the ginsenoside variant flavobacterium DG-16 on the above-ground part of angelica, and the preservation number of the ginsenoside variant flavobacterium DG-16 is CGMCC No.29478.

[0014] Preferably, the dosage of the spraying is (1.5~2.5)×(10 11 ~10 12 ) CFU / acre in terms of the number of viable cells of the ginsenoside variant flavobacterium DG-16, and the spraying period is 1~4 months after the seedling transplanting of angelica, and the spraying is performed once every 4 weeks, and the spraying is performed 3~4 times in total.

[0015] Preferably, the spraying mode comprises foliar spraying.

[0016] Beneficial effects: The application provides a strain of ginsenoside Flavobacterium DG-16 and application of the strain in relieving continuous cropping obstacles of angelica and improving quality of the angelica, and separates a strain of ginsenoside Flavobacterium, which is named as DG-16 and is biologically preserved; the ginsenoside Flavobacterium DG-16 promotes 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 Flavobacterium DG-16 can significantly increase chlorophyll content, 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 relatively significant.

[0017] Biological preservation information The ginsenoside Flavobacterium DG-16 is biologically classified as erigeronis Flavobacterium ginsenosidimutans , and is preserved in the China General Microbiological Culture Collection Center (CGMCC) on January 2, 2024, the preservation address is No. 3, Yikhina Street, Chaoyang District, Beijing, and the preservation number is CGMCC No. 29478. Specific embodiments

[0018] The application provides a strain of ginsenoside Flavobacterium DG-16, and the preservation number of the ginsenoside Flavobacterium DG-16 is CGMCC No. 29478.

[0019] In the application, the ginsenoside Flavobacterium DG-16 is separated from medicinal plants growing in Minxian City, Dingxi City, Gansu Province, China in an alpine region, and is identified as the ginsenoside Flavobacterium through colony characteristics, physical and chemical properties and 16S rDNA sequence identification. The colony characteristics of the ginsenoside Flavobacterium DG-16 on the LB solid culture medium are as follows: yellow-brown, semi-transparent, smooth and moist surface, irregular edge. The 16S rDNA sequence of the ginsenoside Flavobacterium DG-16 is shown in SEQ ID NO: 1.

[0020] The application also provides a microbial agent containing the ginsenoside Flavobacterium DG-16.

[0021] As an implementation manner, the effective viable bacterial concentration of the ginsenoside Flavobacterium DG-16 in the microbial agent is 2x (10 8 ~10 9CFU / mL As an embodiment, the effective component of the bacterial agent can be ginsenoside Bacteroides DG-16, fermentation broth of ginsenoside Bacteroides DG-16, or suspension of ginsenoside Bacteroides DG-16.

[0022] As an embodiment, the preparation method of the fermentation broth of ginsenoside Bacteroides DG-16 comprises the following steps: inoculating the ginsenoside Bacteroides DG-16 into a liquid fermentation medium for fermentation culture to obtain the fermentation broth of ginsenoside Bacteroides DG-16. As an embodiment, the liquid fermentation medium can be PDB medium. As an embodiment, the temperature of the fermentation culture can be 25-30℃, or 28℃. As an embodiment, the time of the fermentation culture can be 1-3d, or 2d. As an embodiment, the shaking frequency of the fermentation culture can be 150-200r / min; as another embodiment, the shaking frequency of the fermentation culture can be 180r / min.

[0023] The present application also provides the use of the ginsenoside Bacteroides DG-16 or the bacterial agent in at least one of improving the quality of angelica, relieving the continuous cropping obstacles of angelica, promoting the growth of angelica, and resisting root rot of angelica.

[0024] As an embodiment, the improvement of the quality of angelica includes the increase of the content of effective components of angelica; as another embodiment, the improvement of the quality of angelica includes the increase of the content of one or more components of ferulic acid, senkyunolide H, ferulic acid coniferyl ester, and ligustilide.

[0025] As an embodiment, the promotion of the growth of angelica includes at least one of the following: 1) increasing the content of endogenous jasmonic acid and / or cytokinin hormones of angelica; 2) reducing the content of gibberellin and / or salicylic acid hormones of angelica; 3) promoting the circulation and utilization of nitrogen; 4) increasing the content of chlorophyll of angelica; 5) increasing at least one of the hundred-straw weight, rhizome length, and stem length of angelica. As an embodiment, the increase of the content of endogenous jasmonic acid and / or cytokinin hormones of angelica includes the increase of the content of one or more hormones of 6-furfurylaminopurine, 2-methylthioisopentenyladenosine riboside, dihydrojasmonic acid, and 12-hydroxyjasmonic acid. As an embodiment, the reduction of the content of gibberellin and / or salicylic acid hormones of angelica includes the reduction of the content of one or more hormones of gibberellin 4, cinnamic acid, and salicylic acid-2-O-β-glucoside.

[0026] The application also provides a method for improving at least one of angelica quality, relieving angelica continuous cropping obstacles, promoting angelica growth and resisting root rot, comprising the following steps: Spraying the bacterial agent containing ginsenoside Brevundimonas variabilis DG-16 on the above-ground part of angelica, wherein the ginsenoside Brevundimonas variabilis DG-16 has a preservation number of CGMCC No.29478.

[0027] As an implementation form, the spraying dose is (1.5-2.5)×(10 11 ~10 12 ) CFU / acre in terms of the viable bacterial count of the ginsenoside Brevundimonas variabilis DG-16; the spraying period is 1-4 months after the angelica seedlings are transplanted, and the spraying is performed once every 4 weeks, and the spraying is performed 3-4 times in total. 6 ~10 7 As an implementation form, the spraying period is 1-4 months after the angelica seedlings are transplanted, and the spraying is performed once every 4 weeks, and the spraying is performed 3-4 times in total; as another implementation form, the spraying is performed 4 times. As an implementation form, the spraying mode comprises foliar spraying. As an implementation form, the bacterial agent in the application can be the fermentation liquor of the ginsenoside Brevundimonas variabilis DG-16, and the fermentation liquor of the ginsenoside Brevundimonas variabilis DG-16 is limited in the above technical solution, and will not be described here.

[0028] The bacterial agent of the ginsenoside Brevundimonas variabilis DG-16 has the functions of nitrogen fixation, siderophore production, nitrification, and high activities of ACC deaminase, cellulase, laccase and catalase. At the same time, the bacterial agent can increase the contents of jasmonic acid and cytokinin, and reduce the contents of gibberellin and salicylic acid; promote the circulation and utilization of nitrogen, significantly increase the content of chlorophyll, and be beneficial to the photosynthesis of angelica; effectively change the physiological and biochemical state of angelica, promote the growth of angelica, relieve the continuous cropping obstacles, and improve the quality.

[0029] In order to further illustrate the application, the technical solutions provided by the application are described in detail below in combination with examples, but they should not be understood as limiting the protection scope of the application.

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

[0031] The reagents and media used in the test were all of chemical purity. The cellulase, laccase, xylanase, peroxidase, ACC deaminase, glutamine synthetase, glutamate synthase, nitrite reductase, nitrate reductase, phenylalanine ammonia lyase activity detection kit, and ABTS, DPPH, siderophore, malondialdehyde, ammonium nitrogen, nitrate nitrogen, amino nitrogen, soluble sugar, chlorophyll, flavonoid, nitrite nitrogen, and nitrate nitrogen content detection kit were purchased from Beijing Boxi Shengong Technology Co., Ltd.

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

[0033] LB medium: tryptone 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% bromo-muskatol blue solution 40 mL / L, adjust pH to 6.8±0.2.

[0037] Glucose peptone water 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] Tryptone ammonium culture medium: tryptone 5 g / L, potassium phosphate dibasic 0.5 g / L, sodium chloride 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, ferrous sulfate 0.01 g / L, pH value is adjusted to 7.2.

[0039] Example 1 The isolation and identification of ginsenoside variant flavobacterium DG-16 are as follows: A strain isolated from medicinal plant angelica grown in Minxian County, Dingxi City, Gansu Province, China by members of the inventor's research group.

[0040] The colony characteristics of the strain are as follows: the isolated strain is inoculated into LB solid culture medium and cultured at 30°C for 2d, and the colony morphology of the strain is obtained as follows: yellow-brown, semi-transparent, smooth and moist surface, and irregular edge.

[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 OD of the sample. 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) Strain enzyme activity determination: ginsenoside variant Flavobacterium DG-16 was inoculated into sterilized 50 mL LB medium, cultured at 30°C with 180 r / min shaking for 2 d, filtered, and the bacterial cells were collected and resuspended in sterile water to a turbidity OD 600 was 1, and the resulting was the test bacterial solution. The test bacterial solution and the enzyme extract were mixed at a volume ratio of 1:10, and the cells were broken by ice-bath ultrasonic treatment. The mixture was centrifuged at 4°C and 8000 r / min for 10 min, and the supernatant was placed on ice for testing. The enzyme activities of peroxidase, cellulase, neutral xylanase, and laccase were determined by spectrophotometry according to the instructions of the enzyme activity kits, and the determination was repeated three times. The determination results were divided by the turbidity of the corresponding bacterial solution, and the enzyme activities were standardized to the enzyme activities of the unit turbidity bacterial solution.

[0055] The results showed that the peroxidase activity of ginsenoside variant Flavobacterium 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: ginsenoside variant Flavobacterium DG-16 was inoculated into sterile glucose peptone water medium using a sterile inoculation loop, and the medium without inoculation was used as a control. The mixture was incubated at 30°C for 2-5 d, 5 drops of methyl red reagent were added to the medium, and the color change was observed. Red color indicated a negative result.

[0057] The results showed that ginsenoside variant Flavobacterium DG-16 could not decompose glucose to produce acid.

[0058] 9) Ammonification test: ginsenoside variant Flavobacterium DG-16 fermentation broth was inoculated into peptone ammonification medium, and six dilution degrees (10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , and 10 -7 ) were used, with three tubes for each dilution degree, and a tube of medium inoculated with sterile water was used as a control. The mixture was cultured in a 30°C constant-temperature incubator. The turbidity of the medium was checked on the 3rd and 5th days after culture. On the 7th day of culture, 5 drops of the culture solution were taken and added to a white porcelain colorimetric plate, and two drops of Nessler's reagent were added to check whether brownish red appeared to determine whether ammonia was produced.

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

[0060] 10) Contact enzyme test: ginsenoside variant Flavobacterium DG-16 colonies were picked and placed on a clean glass slide, and an appropriate amount of 3% hydrogen peroxide solution was added dropwise. A large number of bubbles were generated within 1 min, indicating a positive result.

[0061] The results show that the Flavonoids of Panax Notoginseng-variant Flavobacterium DG-16 has strong catalase activity.

[0062] 11) Strain antagonistic property determination: The antagonistic activity of Flavonoids of Panax Notoginseng-variant Flavobacterium DG-16 and Fusarium solani was determined by plate confrontation culture method: the pathogenic bacteria were inoculated in the center of PDA medium, and the same amount of strain was inoculated on the upper and lower or both sides at the same distance, and cultured at 30℃ for 5-7d, the colony growth and inhibition zone size were observed, and 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 show that Flavonoids of Panax Notoginseng-variant Flavobacterium DG-16 can inhibit the growth of Fusarium solani, and the inhibition rate is 12.47±0.01%.

[0064] Example 3 The influence of Flavonoids of Panax Notoginseng-variant Flavobacterium DG-16 on relieving continuous cropping obstacles and improving quality is as follows: The test groups are set as follows: Treatment group (T): Flavonoids of Panax Notoginseng-variant Flavobacterium DG-16 fermentation broth cultured in PDB medium at 28℃, 180r / min for 2d was diluted 40 times with sterile water to an effective viable bacterial count of 5×(10 6 ~10 7 )CFU / mL. 40L was sprayed per mu.

[0065] Control group (CK): PDB medium without bacteria, diluted with the same multiple as the treatment group, 40L was sprayed per mu.

[0066] The test adopts single factor complete random design, and the land of harvested commodity Angelica sinensis in the previous crop is used to continue Angelica sinensis planting. T and CK are set as two treatments, each with 3 repeats, and the plot area is 30m 2 (4m×7.5m). Angelica sinensis seedlings were transplanted in early April 2024, and foliar spraying was carried out 4 times from May to August, once every 4 weeks. The spraying volume of different plots was equal.

[0067] Field management is carried out according to conventional measures.

[0068] In late August, 5d after the 4th treatment, 100 Angelica sinensis plants were randomly selected from each plot, the leaves were picked, and the rhizosphere soil of the corresponding Angelica sinensis was collected and mixed, and then taken back to the laboratory for standby.

[0069] In late October 2024, the weight of 100 plants, root rot and other growth indicators were counted and determined, 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: Logarithm of fold change with base 2, same below. Inf represents infinity, -Inf represents infinitesimal As shown in Table 3, compared with the control group CK, the hormone content of the treatment group T of the present application has a significant difference. There are a total of 10 significant differences in metabolites, of which 5 are significantly up-regulated and 5 are significantly down-regulated. The treatment of ginsenoside by Amycolatopsis sp. DG-16 increases the content of N-(3-indoleacetyl)-L-phenylalanine, 6-furfurylamino purine, 2-methylthioisopentenyladenosine, dihydro jasmonic acid and 12-hydroxy jasmonic acid, and reduces the content of indole-3-acrylic acid, gibberellin 4, cinnamic acid, salicylic acid-2-O-β-glucoside and 5-deoxyagrostis alcohol hormone. This shows that the bacterial agent of Amycolatopsis sp. can activate the growth-promoting hormones and disease-resistant related hormones of Angelica sinensis, inhibit the salicylic acid hormones and gibberellins, promote growth and improve disease resistance, help to alleviate the continuous cropping obstacles and improve the quality.

[0078] (2) Measurement of leaf physiological and biochemical indexes: The contents of physiological indexes such as ABTS scavenging capacity equivalent value, DPPH scavenging capacity equivalent value, total phenol, malondialdehyde, glutamine synthetase, glutamate synthetase, 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. Each treatment was repeated 3 times, and the results with significant differences and larger values are shown in Table 4.

[0079] Table 4: Measurement of leaf physiological and biochemical indexes (x ± se, n = 3)

[0080] As shown in Table 3, there are significant differences in the related physiological and biochemical indexes of Angelica sinensis leaves, and the contents of glutamate synthase, nitrate reductase, nitrate nitrogen, amino nitrogen and chlorophyll are significantly increased, indicating that the nitrogen assimilation efficiency and photosynthetic pigment synthesis of Angelica sinensis are improved after the treatment of the T group of the present application, which promotes the growth of Angelica sinensis.

[0081] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. A strain of ginsenoside mutant Flavobacterium ( 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. The use of the ginsenoside mutant Flavobacterium DG-16 of claim 1 or the inoculum of any one of claims 2 to 4 in 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.

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

7. The application 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 at least one of the following: weight of 100 Angelica plants, diameter of the rhizome, and length of the plant.

8. A method, said method being at least one of improving the quality of Angelica sinensis, alleviating 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.

9. The method according to claim 8, characterized in that, Based on the viable count of the ginsenoside mutant Flavobacterium DG-16, the spraying dosage is (1.5~2.5)×(10) 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.

10. The method according to claim 8 or 9, characterized in that, The spraying method includes foliar spraying.

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