Bacillus mobilis DG-7 and application thereof in inhibiting bolting of angelica sinensis and improving disease resistance of angelica sinensis

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to bacillus mobilis DG-7 and application thereof in inhibiting bolting of angelica sinensis and improving disease resistance of angelica sinensis. The invention provides a strain of bacillus mobilis DG-7, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.31089. The bacillus mobilis DG-7 and the microbial inoculum thereof provided by the invention can play roles in inhibiting early bolting of angelica sinensis and improving disease resistance by increasing the content of plant endogenous melatonin and salicylic acid, reducing the content of abscisic acid, auxin and gibberellin, reducing the content of total phenolic acid and reducing the expression of photoperiod-related genes. In addition, the bacillus mobilis DG-7 does not solubilize phosphorus, so that the promotion effect of high-concentration phosphorus on early bolting is effectively avoided; the laccase activity of the bacillus mobilis DG-7 is high, polyphenol substances can be effectively removed, and the bolting rate can be reduced.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to a Bacillus mobilis DG-7 and its application in inhibiting the bolting of Angelica sinensis and improving the disease resistance of Angelica sinensis. Background Art

[0002] Angelica sinensis (Oliv.) Diels is an umbelliferous herb, usually distributed in the alpine and rainy shady mountainous areas at an altitude of 1,800 - 3,000 m. Its roots are used as medicine and have the effects of enriching blood and promoting blood circulation, regulating menstruation and relieving pain, and moistening the intestines and relaxing bowel movements. At present, all commercial Angelica sinensis comes from cultivation, and the method of raising seedlings and transplanting is adopted, that is, raising seedlings in the first year, forming medicine in the second year, and reserving seeds in the third year. However, some Angelica sinensis bolts and flowers during the medicine-forming period in the second year and cannot form effective commercial medicinal materials. This phenomenon is called the "early bolting" of Angelica sinensis. After early bolting occurs, the ratio of secondary phloem to secondary xylem in the roots of Angelica sinensis decreases, the parenchyma cells of secondary xylem lignify, the living parenchyma cells and secretory canals decrease, affecting the formation of secondary metabolites, resulting in the lignification and hollowness of fleshy roots, making the roots lack essential oils and losing their medicinal value. In recent years, due to abnormal climate and frequent extreme weather, Angelica sinensis planted in areas with lower altitudes is affected by high temperature and drought, exacerbating the early bolting of Angelica sinensis. In addition, in actual production, in order to relieve soil compaction and promote the growth of the above-ground part, a large amount of phosphate fertilizers such as diamine are applied, resulting in an increase in the bolting rate.

[0003] Although a large number of studies have been carried out on the early bolting problem of Angelica sinensis, there are few inputs that can effectively, simply and low-cost reduce the bolting rate and meet the requirements of GAP. As the "second genome" of plants, microorganisms have significant regulatory effects on plant growth and development, disease resistance, stress resistance, etc. For example, Chinese Patent CN202210228139.6 reports a Bacillus mobilis 1A05942, which has a growth-promoting effect on rape under salt stress conditions; Chinese Patent CN202110203822.X reports a Bacillus mobilis CCTCC NO: M2020863, which can dissociate and release solidified nutrient elements and prepare microbial fertilizers by dissociating coal gangue. However, at present, there is no relevant report on Bacillus mobilis that can inhibit the early bolting of Angelica sinensis and improve the disease control effect. Summary of the Invention

[0004] The purpose of the present invention is to provide a Bacillus mobilis DG-7 and its application in inhibiting the bolting of Angelica sinensis and improving the disease resistance of Angelica sinensis.

[0005] The present invention provides a Bacillus mobilis DG-7 with a preservation number of CGMCC No. 31089.

[0006] The present invention also provides a bacterial agent, which comprises the motile Bacillus DG-7 described in the above technical solution and / or the fermentation broth of the motile Bacillus DG-7 described in the above technical solution.

[0007] Preferably, the concentration of the motile Bacillus DG-7 in the bacterial agent is 2×10 6 ~2×10 9 CFU / mL.

[0008] The present invention also provides the application of the motile Bacillus DG-7 described in the above technical solution or the bacterial agent described in the above technical solution in inhibiting the bolting of Angelica sinensis and / or improving the disease resistance of Angelica sinensis.

[0009] Preferably, the improvement of the disease resistance of Angelica sinensis includes improving the resistance to Angelica root rot.

[0010] The present invention also provides a method for inhibiting the bolting of Angelica sinensis and / or improving the disease resistance of Angelica sinensis, comprising the following steps:

[0011] Two to three months after sowing Angelica sinensis, the seedlings are first foliar sprayed with the bacterial agent described in the above technical solution to be used as Angelica sinensis seedlings;

[0012] The Angelica sinensis seedlings are soaked with the bacterial agent described in the above technical solution and then transplanted, and the transplanted Angelica sinensis seedlings are second foliar sprayed with the bacterial agent described in the above technical solution.

[0013] Preferably, the soaking time is 30-60 min.

[0014] Preferably, the number of times of the first foliar spraying is 2-4 times. Calculated by the viable count of the motile Bacillus DG-7, the dosage of each spraying is 4×10 10 ~8×10 12 CFU / mu.

[0015] Preferably, the number of times of the second foliar spraying is 2-4 times. Calculated by the viable count of the motile Bacillus DG-7, the dosage of each spraying is 4×10 10 ~8×10 12 CFU / mu.

[0016] Preferably, the time interval between two adjacent first foliar sprayings is 2 weeks;

[0017] The time interval between two adjacent second foliar sprayings is 2 weeks.

[0018] Beneficial effects:

[0019] The present invention provides a strain of Bacillus mobilis DG-7 with a preservation number of CGMCC No. 31089. The Bacillus mobilis DG-7 provided by the present invention does not dissolve phosphorus, effectively avoiding the promoting effect of high-concentration phosphorus on early bolting; the Bacillus mobilis DG-7 has strong laccase activity, can effectively remove polyphenolic substances, and helps to reduce the bolting rate. The Bacillus mobilis DG-7 and its bacterial agent of the present invention can inhibit the early bolting of Angelica sinensis and improve disease resistance by increasing the content of endogenous melatonin and salicylic acid in plants, decreasing the content of abscisic acid, auxin, and gibberellin, reducing the total phenolic acid content, and down-regulating the expression of photoperiod-related genes.

[0020] Biological preservation information

[0021] Bacillus mobilis DG-7, with the taxonomic name of Bacillus mobilis, was preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 26, 2024. Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the preservation number of CGMCC No. 31089. Detailed implementation manners

[0022] The present invention provides a strain of Bacillus mobilis DG-7 with a preservation number of CGMCC No. 31089.

[0023] A strain of Bacillus mobilis DG-7 was isolated from a medicinal plant (Angelica sinensis) growing in alpine regions and identified as Bacillus mobilis by colony characteristics and 16S rDNA sequence. The Bacillus mobilis DG-7 of the present invention cannot utilize citrate, does not dissolve phosphorus, has no plate antagonism against pathogenic bacteria such as Fusarium and Alternaria, produces acid (positive in methyl red test), decomposes protein (positive in ammonification test), has strong laccase activity, positive catalase activity, has cellulase, peroxidase, and ACC deaminase activities, has no xylanase activity, and has the ability of nitrification, nitrogen fixation, and siderophore production.

[0024] The present invention also provides a bacterial agent, which includes the Bacillus mobilis DG-7 described in the above technical solution and / or the fermentation broth of the Bacillus mobilis DG-7 described in the above technical solution.

[0025] As an implementation manner, the concentration of Bacillus mobilis DG-7 in the bacterial agent of the present invention is 2×10 6 ~2×10 9 CFU / mL; as another implementation manner, the concentration of Bacillus mobilis DG-7 in the bacterial agent of the present invention is 2×10 7 ~2×10 9CFU / mL; As another embodiment, the concentration of Bacillus cereus DG-7 in the bacterial agent of the present invention is 2×10 8 ~2×10 9 CFU / mL; As another embodiment, the concentration of Bacillus cereus DG-7 in the bacterial agent of the present invention is 5×10 8 ~1×10 9 CFU / mL.

[0026] As one embodiment, the preparation method of the bacterial agent of the present invention includes: inoculating the Bacillus cereus DG-7 into a liquid fermentation medium for fermentation culture to obtain the bacterial agent. As one embodiment, the liquid fermentation medium of the present invention can be a PDB medium. As one embodiment, the temperature of the fermentation culture of the present invention is 25-30°C; as another embodiment, the temperature of the fermentation culture of the present invention is 28°C. As one embodiment, the time of the fermentation culture of the present invention is 1-3 days; as another embodiment, the time of the fermentation culture of the present invention is 2 days. As one embodiment, the oscillation frequency of the fermentation culture of the present invention is 150-200 r / min; as another embodiment, the oscillation frequency of the fermentation culture of the present invention is 180 r / min.

[0027] The Bacillus cereus DG-7 or the bacterial agent of the present invention plays a role in inhibiting the early bolting of Angelica sinensis and improving disease resistance by increasing the content of endogenous melatonin and salicylic acid, decreasing the content of abscisic acid, auxin, and gibberellin, decreasing the total phenolic acid content, and down-regulating the expression of photoperiod-related genes.

[0028] In view of the advantages of the Bacillus cereus DG-7 or the bacterial agent of the present invention, the application of the Bacillus cereus DG-7 or the bacterial agent in inhibiting the bolting of Angelica sinensis and / or improving the disease resistance of Angelica sinensis described in the above technical solution also belongs to the protection scope of the present invention.

[0029] As one embodiment, the improvement of the disease resistance of Angelica sinensis described in the present invention includes improving the resistance to Angelica root rot.

[0030] The present invention also provides a method for inhibiting the bolting of Angelica sinensis and / or improving the disease resistance of Angelica sinensis, including the following steps: 2-3 months after sowing Angelica sinensis, the seedlings are first foliar sprayed with the bacterial agent described in the above technical solution as Angelica sinensis seedlings; the Angelica sinensis seedlings are soaked with the bacterial agent described in the above technical solution and then transplanted, and the transplanted Angelica sinensis seedlings are second foliar sprayed with the bacterial agent described in the above technical solution.

[0031] After sowing Angelica sinensis for 2 to 3 months, the seedlings of Angelica sinensis are first foliar sprayed with the bacterial agent described in the above technical solution to be used as Angelica sinensis seedlings. As an implementation method, seedlings with a diameter of 0.6 to 1 cm at the rhizome part are selected as Angelica sinensis seedlings. As an implementation method, the number of times of the first foliar spraying in the present invention is 2 to 4 times; as another implementation method, the number of times of the first foliar spraying in the present invention is 4 times. As an implementation method, calculated by the viable count of Bacillus subtilis DG-7, the dosage of each first foliar spraying is 4×10 10 ~8×10 12 CFU / mu; as another implementation method, calculated by the viable count of Bacillus subtilis DG-7, the dosage of each first foliar spraying is 4×10 10 ~4×10 12 ; as another implementation method, calculated by the viable count of Bacillus subtilis DG-7, the dosage of each first foliar spraying is 4×10 10 ~4×10 11 . As an implementation method, the bacterial agent described in the above technical solution is diluted 10 to 100 times with water and foliar sprayed at a dosage of 20 to 40 L / mu; as another implementation method, the bacterial agent described in the above technical solution is diluted 100 times with water and foliar sprayed at a dosage of 30 L / mu.

[0032] After obtaining the Angelica sinensis seedlings, the present invention soaks the Angelica sinensis seedlings with the bacterial agent described in the above technical solution to obtain the soaked Angelica sinensis seedlings. As an implementation method, the soaking time in the present invention is 30 to 60 min; as another implementation method, the soaking time in the present invention is 40 to 50 min.

[0033] After obtaining the soaked Angelica sinensis seedlings, the present invention transplants the soaked Angelica sinensis seedlings and performs a second foliar spraying on the transplanted Angelica sinensis seedlings with the bacterial agent described in the above technical solution. As an implementation method, the number of times of the second foliar spraying in the present invention is 2 to 4 times; as another implementation method, the number of times of the second foliar spraying in the present invention is 4 times. As an implementation method, calculated by the viable count of Bacillus subtilis DG-7, the dosage of each second foliar spraying is 4×10 10 ~8×10 12 CFU / mu; as another implementation method, calculated by the viable count of Bacillus subtilis DG-7, the dosage of each second foliar spraying is 4×10 10 ~4×10 12 ; as another implementation method, calculated by the viable count of Bacillus subtilis DG-7, the dosage of each second foliar spraying is 4×10 10 ~4×10 11As an implementation method, the microbial agent described in the above technical solution is diluted 10 to 100 times with water and subjected to a second foliar spraying at a dosage of 20 to 40 L / mu; as another implementation method, the microbial agent described in the above technical solution is diluted 100 times with water and subjected to a second foliar spraying at a dosage of 30 L / mu. As an implementation method, the time interval between two adjacent second foliar sprayings is 2 weeks.

[0034] The present invention does not have special limitations on the process of Angelica sinensis planting and management in the method, and it can be carried out according to the conventional planting and management methods in the art.

[0035] The reagents and culture media used in the experiments of the present invention are all chemically pure. The detection kits for the activities of cellulase, laccase, xylanase, peroxidase, ACC deaminase, glutamine synthetase, glutamate synthase, nitrite reductase, nitrate reductase, and phenylalanine ammonia-lyase, as well as the detection kits for the contents of ABTS, DPPH, siderophore, malondialdehyde, ammonium nitrogen, nitrate nitrogen, amino nitrogen, soluble sugar, chlorophyll, flavonoid, nitrite nitrogen, and nitrate nitrogen are purchased from Beijing Boxbio Science and Technology Co., Ltd. The plant total RNA extraction kit, reverse transcription kit, and SYBR Green fluorescence quantitative kit are purchased from Tiangen Biotech Co., Ltd.

[0036] The components of the culture media used in the examples are as follows:

[0037] PDB medium: 200 g / L of potato, 20 g / L of glucose, natural pH.

[0038] LB medium: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, and the pH value is adjusted to 7.0 - 7.4.

[0039] Modified Stephenson medium: 2 g / L of ammonium sulfate, 0.01 g / L of manganese sulfate, 0.25 g / L of sodium dihydrogen phosphate, 0.03 g / L of magnesium sulfate, 0.5 g / L of calcium carbonate, 0.75 g / L of dipotassium hydrogen phosphate, and the pH value is adjusted to 8.2.

[0040] NBRIP solid medium: 10 g / L of glucose, 5 g / L of calcium phosphate, 5 g / L of magnesium chloride, 0.25 g / L of magnesium sulfate heptahydrate, 0.2 g / L of potassium chloride, 0.1 g / L of ammonium sulfate, 15 g / L of agar, and the pH value is 7.0 ± 0.2.

[0041] Simmon's citrate agar medium: 5.0 g / L of sodium chloride, 0.2 g / L of magnesium sulfate, 1.0 g / L of ammonium dihydrogen phosphate, 1.0 g / L of dipotassium hydrogen phosphate, 5.0 g / L of sodium citrate, 20 g / L of agar, 40 mL / L of 0.2% bromothymol blue solution, and the pH is adjusted to 6.8 ± 0.2.

[0042] Glucose peptone water medium: glucose 0.5 g / L, peptone 0.5 g / L, dipotassium hydrogen phosphate 0.2 g / L, pH adjusted between 7.2 and 7.4.

[0043] Peptone ammonification medium: peptone 5 g / L, dipotassium hydrogen phosphate 0.5 g / L, sodium chloride 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, ferrous sulfate 0.01 g / L, pH adjusted to 7.2

[0044] To further illustrate the present invention, the following examples are used to describe in detail a strain of Bacillus licheniformis DG-7 provided by the present invention and its application in inhibiting the bolting of Angelica sinensis and improving the disease resistance of Angelica sinensis, but they should not be construed as limiting the protection scope of the present invention.

[0045] Example 1

[0046] Isolation and identification of Bacillus licheniformis DG-7

[0047]

[0048] Example 2

[0049] Functional Detection of Bacillus licheniformis DG-7

[0050] 1. Determination of phosphate-solubilizing characteristics: Take 10 μL of the bacterial solution of Bacillus licheniformis DG-7 in Example 1 and inoculate it onto the NBRIP solid medium. Place it in an incubator at a constant temperature of 30°C and culture it. Regularly observe whether a phosphate-solubilizing circle appears on the medium and its size within 7 days. Determine the phosphate-solubilizing ability of the strain according to the size of the phosphate-solubilizing circle of the strain. The results show that Bacillus licheniformis DG-7 does not solubilize phosphate.

[0051] 2. Citrate utilization: Take Bacillus licheniformis DG-7 in Example 1 and inoculate it in the center of the Simmons citrate agar medium with an inoculation loop. Repeat the inoculation 3 times to make the colony distribution uniform. Invert the inoculated plate and culture it in an incubator at a constant temperature of 30°C for 24 - 48 h. Observe whether there is a color change around the colony on the plate. If it turns blue, it is positive. The results show that Bacillus licheniformis DG-7 cannot utilize citrate.

[0052] 3. Plate antagonism experiment: The antibacterial activity of Bacillus licheniformis DG-7 in Example 1 was determined by the plate confrontation method. Specifically: Inoculate the fungal cakes of the pathogenic bacteria strains of Fusarium and Alternaria in the center of the PDA plate respectively. Bacillus licheniformis DG-7 in Example 1 was cross-inoculated 2 cm away from the pathogenic bacteria. Culture it at a constant temperature of 28°C for 7 days. Observe the colony growth status of the pathogenic fungi and the antagonistic bacteria, measure the size of the antibacterial circle with a ruler, and calculate its corresponding antibacterial rate. Antibacterial rate % = (radius of antibacterial circle - radius of antagonistic bacteria) / radius of antagonistic bacteria × 100%. The results show that Bacillus licheniformis DG-7 has no plate antagonism against the pathogenic bacteria strains of Fusarium and Alternaria.

[0053] 4. Nitrification determination: Inoculate Bacillus licheniformis DG-7 in Example 1 into the LB medium and culture it by shaking at 30°C and 180 r / min for 2 days. Centrifuge to collect the bacterial cells and resuspend them in sterile water to an OD 600 of 1.0, and then inoculate it into the sterilized modified Stephenson medium and culture it by shaking at 30°C and 180 r / min for 2 days. Measure the OD 600 value, and detect the content according to the methods described in the nitrite nitrogen and nitrate nitrogen kits. Calculate the nitrification ability using the following formula.

[0054]

[0055] After calculating the nitrification ability, divide the calculation result by the turbidity of the corresponding bacterial solution to standardize it to the nitrification ability of the bacterial solution with a unit turbidity. Repeat each strain 3 times and calculate the average value. The results show that the nitrification ability of the bacterial solution with a unit turbidity of Bacillus licheniformis DG-7 is 1.662 ± 0.35%, indicating a strong nitrification ability.

[0056] 5. Determination of relative content of siderophore: The Bacillus sphaericus DG-7 of Example 1 was inoculated in LB medium, cultured and then centrifuged to obtain the supernatant of siderophore fermentation (denoted as SCS). Two solutions of SCS and chrome azurol S (CAS) were mixed at a volume ratio of 1:1, and the OD of the mixture was measured after incubating in a constant temperature water bath at 37 °C for 0.5 h in the dark. 630 , and the relative content of siderophore of the strain was calculated according to the following formula. The relative content of siderophore of the strain = (Ar - As) / Ar × 100%, where Ar is the OD of the reference substance (the mixture of LB medium and CAS, with the volume ratio of LB medium to CAS being 1:1). 630 ; As is the OD of the sample 630 (the mixture of strain SCS and CAS). The calculation result was divided by the turbidity of the corresponding bacterial solution and standardized to the relative content of siderophore per unit turbidity of the bacterial solution. The results showed that the relative content of siderophore of Bacillus sphaericus DG-7 was 0.592% ± 0.13%.

[0057] 6. Determination of ACC deaminase activity of the strain: The Bacillus sphaericus DG-7 of Example 1 was inoculated in LB medium and cultured with shaking at 30 °C and 180 r / min for 1 d, then centrifuged at 4 °C and 8000 r / min for 10 min, and the supernatant was discarded. The cells were washed twice with DF medium without (NH 4 ) 2 SO 4 , and after centrifugation, the cells were resuspended in ADF medium and cultured at 30 °C and 180 r / min for 1 d. Then, it was centrifuged at 4 °C and 8000 r / min for 10 min, the supernatant was discarded, and the cells were collected. The cells were washed twice with 0.1 mol / L Tris-HCl buffer solution with a pH value of 7.6, centrifuged and resuspended in 0.2 mL of the same concentration Tris-HC1 buffer solution with a pH value of 8.5. A small amount of toluene was added, the cells were broken by ultrasonic treatment, and then 20 μL of 0.5 mol / L 1-aminocyclopropane-1-carboxylic acid (ACC) was added. After mixing, it was incubated in a water bath at 30 °C for 15 min, and then 0.3 mL of dinitrophenylhydrazine was added. The lid was covered and the reaction was carried out in a 30 °C water bath for 0.5 h; then 2 mL of 2 mol / L NaOH was added to terminate the reaction, and the absorbance at 540 nm was measured. The enzyme activity of ACC deaminase was the content of α-ketobutyric acid produced per minute (μmol / min). The results showed that the ACC deaminase activity of Bacillus sphaericus DG-7 was 0.468 ± 0.68 μmol / min.

[0058] 7. Determination of enzyme production activity of the strain: The Bacillus sphaericus DG-7 of Example 1 was inoculated in 50 mL of sterilized LB medium and cultured with shaking at 30 °C and 180 r / min for 2 d, filtered, and the cells were collected and resuspended in sterile water to a turbidity of OD 600It was 1. The test bacterial solution and the enzyme extraction solution were mixed at a volume ratio of 1:10, and the cells were broken by ice-bath ultrasonic treatment. After centrifugation at 8000 r / min for 10 min at 4 °C, the supernatant was placed on ice for further testing. The peroxidase, cellulase, neutral xylanase, and laccase activities were determined by the spectrophotometric method described in the kit instructions, and repeated 3 times. The measurement results were divided by the turbidity of the corresponding bacterial solution to be standardized as the enzyme activity of the bacterial solution per unit turbidity. The results showed that Bacillus motilus DG-7 had no neutral xylanase activity, and the activities of peroxidase, cellulase, and laccase were 1.278 ± 0.296 U / mL, 10.134 ± 0.451 U / mL, and 1.211 ± 0.721 U / mL, respectively. The laccase activity was relatively strong.

[0059] 8. Methyl red test: Using a sterile inoculation loop, inoculate Bacillus motilus DG-7 from Example 1 into a sterile glucose peptone water medium. The non-inoculated medium was used as a control. Incubate at 30 °C for 2 - 5 days, add 5 drops of methyl red reagent to the medium and observe the color change. A red color indicates a positive result. The results showed that Bacillus motilus DG-7 had a strong ability to decompose glucose to produce acid.

[0060] 9. Ammonification test: Inoculate the fermentation broth of Bacillus motilus DG-7 from Example 1 (OD 600 was 1.5 - 1.8) into a peptone ammonification medium. Six dilutions (10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 ) were inoculated, and 3 tubes were inoculated for each dilution. Another tube of the medium was inoculated with sterile water as a control. Incubate in a constant temperature incubator at 30 °C. Check the turbidity of the medium on the 3rd and 5th days after incubation. On the 7th day of incubation, pipette 5 drops of the culture solution onto a white porcelain colorimetric plate, add 2 drops of Nessler's reagent, and check whether a brownish color appears to determine whether ammonia is produced. The results showed that Bacillus motilus DG-7 had a strong ammonification ability, indicating its relatively strong protein decomposition ability.

[0061] 10. Catalase test: Pick a colony of Bacillus motilus DG-7 from Example 1 and place it on a clean glass slide. Add an appropriate amount of 3% hydrogen peroxide solution. If bubbles are produced within 1 min, it is a positive result, and the strength can be preliminarily judged according to the amount of bubbles. The results showed that Bacillus motilus DG-7 had strong catalase activity.

[0062] 11. Degradation of autotoxic substances: Using de - sugared M9 medium as the basal medium, 50 mg / L of ferulic acid and 50 mg / L of anthraquinone were added as the sole carbon sources respectively. The Bacillus sphaericus DG - 7 in Example 1 was inoculated into the above - mentioned media and M9 medium respectively. The inoculated media were cultured at 30 °C and 180 r / min for 3 - 5 days, and the growth status in the two media was compared, and the OD 600 value was measured. The results showed that: Bacillus sphaericus DG - 7 could degrade ferulic acid but could not degrade anthraquinone.

[0063] Example 3

[0064] Bacillus sphaericus DG - 7 inhibits premature bolting of Angelica sinensis

[0065] 1. Preparation of treatment solution

[0066] The Bacillus sphaericus DG - 7 in Example 1 was inoculated into PDB medium and cultured with shaking at 28 °C and 180 r / min for 2 days, and then diluted with water to an effective viable bacteria count of 2×10 6 ~2×10 7 CFU / mL to obtain the treatment solution of Bacillus sphaericus DG - 7; PDB medium without bacteria was taken and diluted by the same multiple as the treatment solution of Bacillus sphaericus DG - 7 to obtain the control treatment solution.

[0067] 2. Using Angelica sinensis seeds as experimental materials, the variety is Min Gui No. 1. The experiment was carried out at the Chinese medicinal materials planting base of Tianzhu Green Energy Agricultural Technology Co., Ltd. from April to October in 2023 and 2024. The soil type is chestnut soil, and the pH value is 8.5; the land was prepared at the end of April, and organic fertilizer (organic matter ≥ 45%, total nutrients N + P 2 O 5 +K 2 O ≥ 4%) 1000 kg / mu was applied. A single - factor completely randomized design was used for the experiment, with 2 treatments, namely the treatment group (T) and the control group (CK), and each treatment had 3 replicates. The plot area was 30 m 2 (4 m × 7.5 m).

[0068] Angelica sinensis was sown in mid - June 2023, and the leaves were sprayed 4 times from August to September, once every 2 weeks. The spraying volume in different plots was equal. Among them, when spraying in the treatment group (T) each time, 20 L of the treatment solution of Bacillus sphaericus DG - 7 was sprayed per mu of land; when spraying in the control group (CK) each time, 20 L of the control treatment solution was sprayed per mu of land. The seedlings were dug up at the beginning of October 2023 and stored by conventional methods.

[0069] In April 2024, seedlings with a diameter of 0.6 - 1 cm at the rhizome part were selected as Angelica sinensis seedling stocks. (For Angelica sinensis seedlings with a rhizome diameter of less than 0.6 cm, in areas such as Tianzhu where the early spring is low in temperature and heavy in drought, the emergence rate is low and the growth is slow due to environmental influence, which has a greater impact on the production of commercial medicinal materials). The corresponding Angelica sinensis seedling stocks were soaked in the treatment solutions used in the treatment group (T) and the control group (CK) for 30 minutes respectively, and after draining the surface moisture, they were transplanted. After emergence, the corresponding treatment solutions were sprayed on the leaves of Angelica sinensis (without film mulching), once every 2 weeks for a total of 4 times, and the spraying volume in different plots was equal. Field management was carried out according to conventional measures.

[0070] At the end of August 2024, the number of bolting plants and the number of root rot disease plants were counted, and the early bolting inhibition rate and disease inhibition rate were calculated according to the following formulas. The results are shown in Table 1.

[0071] Early bolting inhibition rate = (bolting rate of the control group - bolting rate of the treatment group) / bolting rate of the control group × 100%;

[0072] Disease inhibition rate = (disease rate of the control group - disease rate of the treatment group) / disease rate of the control group × 100%.

[0073] Table 1 Effects of bacterial agents on early bolting and root rot of Angelica sinensis

[0074] Treatment method Bolting rate / % Early bolting inhibition rate / % Disease rate / % Disease inhibition rate / % T 44.30±1.05b 50.19 15±3b 50 CK 88.94±1.72a - 30±2a -

[0075] Note: The lowercase letters in the table indicate P < 0.05.

[0076] It can be seen from Table 1 that after treating Angelica sinensis with Bacillus licheniformis DG - 7, the bolting rate was 44.30%, and the early bolting inhibition rate was 50.19%. Bacillus licheniformis DG - 7 can significantly inhibit the early bolting of Angelica sinensis and reduce the incidence of root rot.

[0077] Example 4

[0078] Effects of Bacillus licheniformis DG - 7 on the endogenous hormone levels of Angelica sinensis seedlings

[0079] 1. Preparation of treatment solution

[0080] Inoculate Bacillus licheniformis DG - 7 from Example 1 into PDB medium, and culture it with shaking at 28°C and 180 r / min for 2 days, then dilute it with water to an effective viable bacteria count of 2×10 6 ~2×10 7 CFU / mL to obtain the treatment solution of Bacillus licheniformis DG - 7; Take PDB medium without adding bacteria and dilute it by the same multiple as the treatment solution of Bacillus licheniformis DG - 7 to obtain the control treatment solution.

[0081] 2. Using Angelica sinensis seeds as experimental materials, the variety is Min Gui No. 1. The experiment was conducted from April to October in 2023 and 2024 at the Chinese herbal medicine planting base of Tianzhu Green Energy Agricultural Technology Co., Ltd. The soil type is chestnut soil with a pH value of 8.5. The land was prepared at the end of April, and organic fertilizer (organic matter ≥ 45%, total nutrients N + P 2 O 5 +K 2 O ≥ 4%) was applied at 1000 kg per mu. The experiment was carried out using a single-factor completely randomized design, with 2 treatments, namely the treatment group (DG-7) and the control group (CK), and each treatment had 3 replicates. The plot area was 30 m 2 (4 m × 7.5 m).

[0082] Angelica sinensis was sown in mid-June 2023, and the foliar spraying was carried out 4 times from August to September, once every 2 weeks. The spraying volume in different plots was equal. Among them, when spraying in the treatment group (DG-7) each time, 20 L of the treatment solution of Bacillus licheniformis DG-7 was sprayed per mu of land; when spraying in the control group (CK) each time, 20 L of the control treatment solution was sprayed per mu of land. Three days after the 4th treatment, 30 Angelica sinensis seedlings were randomly selected from each plot, and the leaves of the same part were picked, mixed respectively, and stored in liquid nitrogen for later use.

[0083] 3. Endogenous hormone detection: Ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS) was used to determine the endogenous hormones in Angelica sinensis seedlings (the contents of free abscisic acid and conjugated abscisic acid (abscisic acid glucoside) were detected, and the sum of the two was calculated and defined as total abscisic acid; the contents of indole-3-acetonitrile, indoleacetic acid-valine methyl ester, indole-3-acetic acid, indole-3-acetic acid methyl ester, and indoleacetic acid-aspartic acid were detected, and the sum of the aforementioned auxins was calculated and defined as total auxin; the contents of 2-methylthio-isopentenyladenosine, isopentenyladenosine, N6-isopentenyladenine, dihydrozeatin-7-glycoside, trans-zeatin-9-glycoside-5'-monophosphate, N-6-isopentenyladenosine 5'-monophosphate, trans-zeatin-9-β-glucoside, dihydrozeatin nucleoside-O-glycoside, 6-furfurylaminopurine, and zeatin riboside were detected, and the sum of the aforementioned cytokinins was calculated and defined as total cytokinin; the contents of gibberellin 3, precursor 5 of gibberellin 3, gibberellin 1, precursor 20 of gibberellin 1, gibberellin 7, gibberellin 4, gibberellin 4, and precursor 9 of gibberellin 4,7 were detected, and the sum of the aforementioned gibberellins was calculated and defined as total gibberellin, and the sum of gibberellin 1, 3, 4, and 7 was calculated and defined as active gibberellin; the contents of 12-hydroxyjasmonic acid, 12-oxo-phytodienoic acid, methyl jasmonate, jasmonic acid-isoleucine, and jasmonic acid were detected, and the sum of the aforementioned substances was calculated and defined as total jasmonic acid; the contents of salicylic acid-2-O-β-glucoside and methyl salicylate O-β-glucoside were detected, and the sum of the two was calculated and defined as conjugated salicylic acid; free salicylic acid, free salicylic acid precursor o-hydroxycinnamic acid, precursor of ethylene 1-aminocyclopropane carboxylic acid (ACC), and melatonin content were detected; the fold change (FC, mean value of treatment group / mean value of control group) and log2(FC) value were statistically calculated, and the results are shown in Table 2.

[0084] Table 2 Endogenous hormone contents in Angelica sinensis seedlings (x±se, n = 3)

[0085] Index DG-7 CK FC log2(FC) Total abscisic acid (ng / g) 128.309±1.195b 182.399±1.957a 0.703 -0.507 Free abscisic acid (ng / g) 66.101±0.586b 157.738±2.825a 0.419 -1.255 Bound abscisic acid (ng / g) 62.208±0.613a 24.662±1.092b 2.522 1.334 Total auxin (ng / g) 24.315±1.317b 37.723±1.066a 0.644 -0.633 Total cytokinin (ng / g) 45.476±0.945a 32.765±1.072b 1.387 0.472 ACC (ng / g) 0 0 - - Active gibberellin (ng / g) 0 0 - - Total gibberellin (ng / g) 14.626±0.093a 4.091±0.387b 3.571 1.836 Total jasmonic acid (ng / g) 1356.681±4.849b 1804.186±20.274a 0.752 -0.411 Melatonin (ng / g) 0.117±0.003a 0.149±0.005a - - o-Hydroxycinnamic acid (ng / g) 131.65±0.866a 108.177±2.435b 1.217 0.283 Free salicylic acid (ng / g) 92.838±1.349a 72.724±2.239b 1.276 0.352 Bound salicylic acid (ng / g) 373.511±4.289b 399.440±3.369a 0.935 -0.096

[0086] Note: Lowercase letters in the table indicate P < 0.05.

[0087] As can be seen from Table 2, the contents of ACC and active gibberellin are zero, and the melatonin content is very low and there is no significant difference. Compared with the control, there are significant differences in the contents of other hormones in the DG-7 treatment group: the DG-7 treatment reduced the content of free abscisic acid to a large extent, and the value of log2(FC) was -1.255; it increased the content of inactive conjugated abscisic acid to a large extent, and the value of log2(FC) was 1.334, and the total abscisic acid content decreased; the total content of auxin decreased; the total content of cytokinin increased; the total content of jasmonic acid decreased; free salicylic acid and the salicylic acid precursor o-hydroxycinnamic acid increased, while conjugated salicylic acid decreased, but the change degree was small. To sum up, the treatment with Bacillus licheniformis DG-7 significantly reduced the contents of abscisic acid and auxin, increased the content of free salicylic acid, inhibited seedling growth, reduced root branching, enhanced biotic stress resistance, helped reduce the bolting rate, and improved disease resistance.

[0088] Example 5

[0089] Effect of Bacillus licheniformis DG-7 on the endogenous physiological and biochemical status and expression of bolting-related genes of transplanted Angelica sinensis

[0090] 1. Preparation of treatment solution

[0091] Inoculate Bacillus licheniformis DG-7 from Example 1 into PDB medium, shake culture at 28 °C and 180 r / min for 2 days, and dilute it with water to an effective viable bacteria count of 2×10 6 ~2×10 7 CFU / mL to obtain the treatment solution of Bacillus licheniformis DG-7; take PDB medium without bacteria and dilute it by the same multiple as the treatment solution of Bacillus licheniformis DG-7 to obtain the control treatment solution.

[0092] 2. Use the one-year-old Angelica sinensis seedlings (the diameter of the rhizome part is 0.6 - 1 cm) corresponding to Example 4 as the test materials. The test was carried out at the Chinese medicinal materials planting base of Tianzhu Green Energy Agricultural Technology Co., Ltd. from April to October 2024. The soil type is chestnut soil, and the pH value is 8.5; the land was prepared at the end of April, and organic fertilizer (organic matter ≥ 45%, total nutrients N+P 2 O 5 +K 2 O≥4%) 1000 kg / mu was applied. The experiment was carried out using a single-factor completely randomized design, with 2 treatments: the treatment group (DG-7) and the control group (CK), with 3 replicates for each treatment, and the plot area was 30 m 2 (4 m × 7.5 m).

[0093] Before transplanting the one-year-old Angelica sinensis seedlings in the treatment group (DG-7), soak them in the treatment solution of Bacillus licheniformis DG-7 for 30 min; before transplanting the one-year-old Angelica sinensis seedlings in the control group (CK), soak them in the control treatment solution for 30 min; drain the surface moisture and transplant. After emergence, spray the treatment solution of Bacillus licheniformis DG-7 on the leaves of Angelica sinensis in the treatment group (DG-7) once every two weeks for a total of 4 times, with a spraying amount of 20 L / mu each time; spray the control treatment solution on the leaves of Angelica sinensis in the control group (CK) once every two weeks for a total of 4 times, with a spraying amount of 20 L / mu each time. Three days after the fourth treatment of the treatment group (DG-7) and the control group (CK), randomly select 10 Angelica sinensis plants in each plot, cut the functional leaves at the same position of the 3rd to 4th positions, mix the samples respectively, and store them in liquid nitrogen for later use.

[0094] 3. Determination of physiological and biochemical indexes: According to the kit instructions, use spectrophotometry to determine the activities of glutamine synthetase, glutamate synthase, nitrite reductase, nitrate reductase, phenylalanine ammonia-lyase and the contents of ABTS, DPPH, total phenols, malondialdehyde, ammonium nitrogen, nitrate nitrogen, amino nitrogen, soluble sugar, chlorophyll, and flavonoids in Angelica sinensis leaves. Each treatment was repeated 3 times, and the results are shown in Table 3.

[0095] Table 3 Detection results of physiological and biochemical indexes during the cultivation period of Angelica sinensis (x±se, n = 3)

[0096] Index DG-7 CK FC log2(FC) ABTS scavenging capacity equivalent quantification value (mmol / L) 0.788±0.012a 0.890±0.034a - - DPPH scavenging capacity equivalent quantification value (mmol / L) 0.992±0.011a 1.012±0.016a - - Total phenol (mg / g) 1.189±0.008b 1.391±0.028a 0.855 -0.225 Malondialdehyde (nmol / g) 20.384±0.591a 14.611±0.203b 1.395 0.480 Glutamine synthetase (U / g) 9.398±0.336a 8.317±0.063b 1.130 0.176 Glutamate synthase (U / g) 92.163±3.215a 84.663±2.663b 1.088 0.122 Nitrite reductase (U / g) 3.189±0.096a 3.383±0.103a - - Nitrate reductase (U / g) 1.757±0.607a 2.690±0.401a - - Ammonium nitrogen (μg / g) 64.494±0.626b 73.103±0.723a 0.882 -0.181 Nitrate nitrogen (μg / g) 18.838±2.285a 20.776±1.814a - - Amino nitrogen (μg / g) 42.426±1.655b 71.660±0.540a 0.592 -0.756 Total nitrogen (μg / g) 124.240±0.388b 165.330±0.546a 0.751 -0.412 Soluble sugar (mg / g) 22.584±0.191a 22.026±0.124a - - Chlorophyll (mg / g) 5.708±0.039b 6.013±0.044a 0.825 -0.276 PAL (U / g) 105.068±6.146b 131.886±0.832a 0.796 -0.327 Flavonoid (mg / g) 0.404±0.014b 0.530±0.005a 0.763 -0.389

[0097] Note: Lowercase letters in the table indicate P < 0.05.

[0098] It can be seen from Table 3 that the treatment with Bacillus licheniformis DG-7 has no significant effect on the scavenging of reactive oxygen species, significantly increases the contents of glutamine synthetase and glutamate synthase, and decreases the contents of amino nitrogen and total nitrogen, indicating that Bacillus licheniformis DG-7 has a certain inhibitory effect on the growth of Angelica sinensis; after the treatment with Bacillus licheniformis DG-7, the PAL activity decreases, and the contents of total phenols and flavonoids decrease, indicating that the treatment with Bacillus licheniformis DG-7 has the effect of reducing the content of total phenolic acid compounds.

[0099] 4. Detection of endogenous hormones: Use ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS) to determine the content of endogenous hormones in Angelica sinensis leaves. The detection indexes are the same as in Example 4, and the results are shown in Table 4.

[0100] Table 4 Content of endogenous hormones during the cultivation period of Angelica sinensis (x±se, n = 3)

[0101]

[0102]

[0103] Note: Lowercase letters in the table indicate P < 0.05.

[0104] As can be seen from Table 4, except for conjugated abscisic acid, there are significant differences in other indicators between the DG-7 and CK treatment groups. Compared with the CK treatment group, after treatment with strain DG-7, the melatonin content increased significantly and to a large extent, with the log2(FC) value being 1.975; the conjugated salicylic acid content increased significantly and to a large extent, with the log2(FC) value being 3.204. The total abscisic acid and free abscisic acid contents decreased significantly, which was consistent with the performance in the seedling stage; the auxin content decreased, and the cytokinin content increased, which was consistent with the performance in the seedling stage; the ACC content increased; the total content of active gibberellin and gibberellin was significantly higher than that in the seedling stage, and the content decreased significantly after the treatment with the bacterial agent; the total jasmonic acid decreased significantly, which was consistent with the performance in the seedling stage; the free salicylic acid and its precursors, and the conjugated salicylic acid content increased significantly. In summary, the treatment with Bacillus sphaericus DG-7 mainly reduced the bolting rate by increasing the endogenous melatonin and salicylic acid contents, and decreasing the abscisic acid, auxin, and gibberellin contents.

[0105] 5. Detection of the expression levels of genes related to bolting

[0106] (1) RNA extraction: Grind the Angelica sinensis samples into powder rapidly in liquid nitrogen, add an appropriate amount of lysis buffer and immediately vortex and mix evenly, transfer to the filter column CS, centrifuge at 12000 r / min for 2 min, aspirate the supernatant and add 0.5 times the volume of absolute ethanol, mix evenly and transfer to the adsorption column CR3, and perform the subsequent operations according to the instructions. Elute with 50 μL of RNase-Free ddH 2 O to obtain RNA, and store it at -80 °C.

[0107] (2) Reverse transcription: Reaction system (20 μL): 4 μL of 5×FastKing-RT SuperMix, 1 μg of RNA, and make up to 20 μL with RNase-Free ddH 2 O; Reaction program: 42 °C for 15 min (to remove genomic DNA and perform reverse transcription reaction), 95 °C for 3 min (enzyme inactivation process) to obtain the cDNA template.

[0108] (3) qRT-PCR Refer to the key genes in different pathways related to the bolting of Angelica sinensis (Li Jie. Research on the regulatory mechanism of bolting and flowering of Angelica sinensis in the photoperiod stage [D]. Gansu Agricultural University, 2021. DOI: 10.27025 / d.cnki.ggsnu.2021.000082.), and the primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The detailed sequences are shown in Table 5. Each sample was repeated 3 times, and the average value was taken. The results of the relative expression levels are shown in Table 6.

[0109] Table 5 qRT-PCR primer sequences

[0110]

[0111]

[0112] Table 6 Detection results of gene expression levels (x±se, n = 3)

[0113] Index DG-7 CK log2(FC) GAI 3.458±0.418a 1.022±0.144b 1.758 GA20OX1 0.149±0.029b 1.044±0.198a -2.805 GA2OX6 0.031±0.007b 1.004±0.059a -5.017 GA2OX8 0.481±0.111b 1.010±0.076a -1.071 AP1 0.047±0.002b 1.003±0.026a -4.415 SOC1 0.199±0.003b 1.003±0.049a -2.331 HD3A 0.084±0.003b 1.007±0.084a -3.584 MASD8 0.695±0.023b 1.005±0.069a -0.531 CO3 0.222±0.029b 1.027±0.159a -2.211 AGL8 0.943±0.377a 1.002±0.026a - FD 0.044±0.007b 1.010±0.076a -4.523

[0114] Note: Lowercase letters in the table indicate P < 0.05.

[0115] It can be seen from Table 6 that the indicators with the absolute value of log2(FC) greater than 1 are GAI, GA20OX1, GA2OX6, GA2OX8, AP1, SOC1, HD3A, CO3, and FD. GAI is a negative regulatory gene for gibberellin synthesis, and its expression was significantly up-regulated after treatment with the bacterial agent. GA20OX1, GA2OX6, and GA2OX8, as genes encoding gibberellin synthesis, had significantly down-regulated expression levels, which was consistent with the decrease in the content of the physical and chemical index gibberellin. The photoperiod pathway genes HD3A, CO3, and FD and the integration gene SOC1 are all key positive regulatory genes related to bolting. After treatment with the bacterial agent, their expression levels were all significantly down-regulated, indicating that: the treatment with Bacillus motile DG-7 can affect the photoperiod and play a role in inhibiting the early bolting of Angelica sinensis.

[0116] It can be seen from the above content that the Bacillus motile DG-7 provided by the present invention can inhibit the early bolting of Angelica sinensis, reduce the bolting rate, and improve the disease resistance of Angelica sinensis, especially reduce the incidence of Angelica sinensis root rot.

[0117] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus mobilis DG-7, with a deposit number of CGMCC No.31089.

2. A bacterial agent, characterized in that The bacterial agent includes the Bacillus mobilis DG-7 according to claim 1 and / or the fermentation broth of the Bacillus mobilis DG-7 according to claim 1.

3. The bacterial agent according to claim 2, characterized in that The concentration of Bacillus mobilis DG-7 in the bacterial agent is 2×10 6 ~2×10 9 CFU / mL.

4. Use of the Bacillus mobilis DG-7 according to claim 1 or the bacterial agent according to claim 2 or 3 in inhibiting the bolting of Angelica sinensis and / or improving the disease resistance of Angelica sinensis.

5. The use according to claim 4, characterized in that: The improving disease resistance of Angelica sinensis includes improving the resistance of Angelica sinensis to root rot.

6. A method for inhibiting the bolting of Angelica sinensis and / or improving the disease resistance of Angelica sinensis, characterized in that: The steps include: 2 to 3 months after the angelica is sown, the seedlings are first sprayed with the bacterial agent described in claim 2 or 3 to obtain angelica seedlings; The angelica seedlings are soaked in the microbial agent described in claim 2 or 3 and then transplanted, and the transplanted angelica seedlings are sprayed with the microbial agent described in claim 2 or 3 for a second time on the leaves.

7. The method according to claim 6, characterized in that The soaking time is 30 to 60 minutes.

8. The method according to claim 6, characterized in that The number of times of the first leaf spraying is 2 to 4 times, and the dosage of each spraying is 4×10 10 ~8×10 12 CFU / acre.

9. The method according to claim 6, characterized in that The second leaf spraying was performed 2 to 4 times, and the dosage for each spraying was 4×10 10 ~8×10 12 CFU / acre.

10. The method according to any one of claims 6 to 9, characterized in that: The time interval between two adjacent first foliar sprayings is 2 weeks; The time interval between two adjacent second foliar sprayings is 2 weeks.

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