Bacillus sp. DG-7 and application thereof in inhibiting angelica archangelica bolting and improving disease resistance of angelica archangelica

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a bacillus sp. DG-7 and application of the bacillus sp. DG-7 in inhibiting angelica archangelica bolting and improving disease resistance of the angelica archangelica. The application provides the bacillus sp. DG-7 with a preservation number of CGMCC No. 31089. The bacillus sp. DG-7 and the bacterial agent thereof can increase endogenous melatonin and salicylic acid content, reduce abscisic acid, auxin and gibberellin content, reduce total phenolic acid content, and down-regulate expression of a photoperiod-related gene, so as to inhibit early bolting of the angelica archangelica and improve disease resistance. In addition, the bacillus sp. DG-7 does not decompose phosphorus, so that the promoting effect of high-concentration phosphorus on early bolting is effectively avoided; the bacillus sp. DG-7 has strong laccase activity, can effectively remove polyphenols, and is helpful to reduce the bolting rate.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a Bacillus mobilis DG-7 and application thereof in inhibiting angelica bolting and improving disease resistance of angelica. BACKGROUND

[0002] Angelica (Angelica sinensis (Oliv.) Diels) is a perennial herb of the family Umbelliferae, which is usually distributed in the high-cold and rainy mountainous area with an altitude of 1800-3000 m. The root of angelica is used as medicine, which has the effects of tonifying blood, activating blood, regulating menstruation, relieving pain, moistening the intestines, and so on. At present, commercial angelica is obtained by cultivation, that is, seedlings are grown in the first year, and the roots are harvested in the second year, and seeds are reserved in the third year. However, some angelica bolts and flowers in the second year, which cannot form effective commercial medicinal materials. This phenomenon is called “early bolting” of angelica. After early bolting occurs, the ratio of secondary phloem and secondary xylem in the root of angelica decreases, the parenchyma cells of the secondary xylem are lignified, the living parenchyma cells and secretory ducts decrease, which affects the formation of secondary products, causes the xylem of the fleshy root to be lignified and hollow, and makes the root lack oil and gas, thereby losing medicinal value. In recent years, due to abnormal climate and frequent extreme weather, the angelica planted in low-altitude areas is affected by high temperature and drought, which aggravates the early bolting of angelica. In addition, in actual production, in order to alleviate soil compaction and promote the growth of the aboveground part, a large amount of diamine and other phosphorus fertilizers are applied, which leads to an increase in the bolting rate.

[0003] Although a large number of studies have been carried out on the early bolting problem of angelica, there are few effective, simple, low-cost inputs that can reduce the early bolting rate and meet the requirements of GAP. Microorganisms, as the “second genome” of plants, have a significant regulatory effect on the growth and development, disease resistance, and stress resistance of plants. For example, Chinese patent CN202210228139.6 reports a Bacillus mobilis 1A05942 which has a growth-promoting effect on oilseed rape under salt stress; Chinese patent CN202110203822.X reports a Bacillus mobilis CCTCC NO: M2020863 which can dissociate and release solidified nutrient elements and prepare a microbial fertilizer by dissociating coal gangue. However, there is no relevant report on a Bacillus mobilis which can inhibit the early bolting of angelica and improve the disease control effect. SUMMARY

[0004] The present application aims to provide a Bacillus mobilis DG-7 and application thereof in inhibiting angelica bolting and improving disease resistance of angelica.

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

[0006] The application further provides a bacterial agent, which comprises the Bacillus sp. DG-7 and / or the fermentation liquor of the Bacillus sp. DG-7.

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

[0008] The application further provides the use of the Bacillus sp. DG-7 or the bacterial agent in inhibiting the bolting of Angelica sinensis and / or improving the disease resistance of the Angelica sinensis.

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

[0010] The application further provides a method for inhibiting the bolting of Angelica sinensis and / or improving the disease resistance of the Angelica sinensis, which comprises the following steps:

[0011] After the Angelica sinensis is sowed for 2-3 months, the bacterial agent is used for the first time to spray the seedlings, and the seedlings are used as the Angelica sinensis seedlings.

[0012] After the Angelica sinensis seedlings are soaked in the bacterial agent, the bacterial agent is used to spray the transplanted Angelica sinensis seedlings.

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

[0014] Preferably, the first time of spraying is 2-4 times, and the dosage of each spraying is 4×10 10 ~ 8×10 12 CFU / mu.

[0015] Preferably, the second time of spraying is 2-4 times, and the dosage of each spraying is 4×10 10 ~ 8×10 12 CFU / mu.

[0016] Preferably, the interval between the two adjacent times of the first spraying is 2 weeks.

[0017] The interval between the two adjacent times of the second spraying is 2 weeks.

[0018] Beneficial effects:

[0019] This invention provides a strain of Bacillus mobilis, DG-7, with accession number CGMCC No. 31089. The Bacillus mobilis DG-7 provided by this invention does not solubilize phosphorus, effectively avoiding the promoting effect of high concentrations of phosphorus on premature bolting. The Bacillus mobilis DG-7 has strong laccase activity, which can effectively remove polyphenolic substances, helping to reduce bolting rate. The Bacillus mobilis DG-7 and its inoculum agent described in this invention can inhibit premature bolting of Angelica sinensis and improve disease resistance by increasing the content of endogenous melatonin and salicylic acid in plants, reducing the content of abscisic acid, auxin, and gibberellin, reducing the total phenolic acid content, and downregulating the expression of photoperiod-related genes.

[0020] Biological Preservation Information

[0021] Bacillus mobilis DG-7 was deposited on June 26, 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 CGMCC No. 31089. Detailed Implementation

[0022] This invention provides a strain of Bacillus mobilis DG-7, with accession number CGMCCNo.31089.

[0023] This invention isolates a strain of *Bacillus motility* DG-7 from a medicinal plant (Angelica sinensis) growing in high-altitude and cold regions. It was confirmed as *Bacillus motility* through colony characteristics and 16S rDNA sequence identification. The *Bacillus motility* DG-7 described in this invention cannot utilize citrate, does not solubilize phosphorus, has no plate antagonistic effect against pathogens such as *Fusarium* and *Alternaria*, produces acid (positive methyl red test), decomposes proteins (positive ammonification test), has strong laccase activity, is positive for catalase activity, and exhibits cellulase, peroxidase, and ACC deaminase activities, but lacks xylanase activity. It possesses nitrification, nitrogen fixation, and heparin-producing abilities.

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

[0025] In one embodiment, the concentration of Bacillus motility DG-7 in the bacterial agent of the present invention is 2 × 10⁻⁶. 6 ~2×10 9 CFU / mL; as another embodiment, the concentration of Bacillus motility DG-7 in the bacterial agent of the present invention is 2 × 10⁻⁶ CFU / mL. 7 ~2×10 91×105CFU / mL; as another embodiment, the concentration of Bacillus sp. DG-7 in the bacterial agent is 2×105CFU / mL. 8 1×105CFU / mL; as another embodiment, the concentration of Bacillus sp. DG-7 in the bacterial agent is 2×105CFU / mL. 9 1×105CFU / mL; as another embodiment, the concentration of Bacillus sp. DG-7 in the bacterial agent is 2×105CFU / mL. 8 1×105CFU / mL; as another embodiment, the concentration of Bacillus sp. DG-7 in the bacterial agent is 2×105CFU / mL. 9 1×105CFU / mL; as another embodiment, the concentration of Bacillus sp. DG-7 in the bacterial agent is 2×105CFU / mL.

[0026] As an embodiment, the preparation method of the bacterial agent comprises: inoculating Bacillus sp. DG-7 into a liquid fermentation medium for fermentation culture to obtain the bacterial agent. As an embodiment, the liquid fermentation medium can be PDB medium. As an embodiment, the fermentation culture temperature is 25-30℃; as another embodiment, the fermentation culture temperature is 28℃. As an embodiment, the fermentation culture time is 1-3d; as another embodiment, the fermentation culture time is 2d. As an embodiment, the fermentation culture shock frequency is 150-200r / min; as another embodiment, the fermentation culture shock frequency is 180r / min.

[0027] The Bacillus sp. DG-7 or the bacterial agent can inhibit the early bolting of Angelica sinensis, improve the disease resistance of Angelica sinensis by increasing the content of endogenous melatonin and salicylic acid, reducing the content of abscisic acid, auxin and gibberellin, and down-regulating the expression of photoperiod-related genes.

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

[0029] As an embodiment, the method for improving the disease resistance of Angelica sinensis comprises improving the resistance of Angelica sinensis to root rot.

[0030] The present application also provides a method for inhibiting the bolting of Angelica sinensis and / or improving the disease resistance of Angelica sinensis, comprising the following steps: after Angelica sinensis is sown for 2-3 months, the bacterial agent is used for the first leaf surface spraying of seedlings, and the bacterial agent is used for soaking the seedlings before transplanting, and the bacterial agent is used for the second leaf surface spraying of the transplanted seedlings.

[0031] The application utilizes the bacterial agent of the above technical solution to carry out first leaf surface spraying on the angelica seedling after the angelica is sowed for 2-3 months, and the angelica seedling is used as the angelica seedling. As an embodiment, the seedling with the diameter of 0.6-1 cm at the base of the stem is selected as the angelica seedling. As an embodiment, the first leaf surface spraying is carried out for 2-4 times. As another embodiment, the first leaf surface spraying is carried out for 4 times. As an embodiment, the dose of the first leaf surface spraying is 4×10 10 ~8×10 12 CFU / mu according to the number of viable bacteria of Bacillus circulans DG-7. As another embodiment, the dose of the first leaf surface spraying is 4×10 10 ~4×10 12 CFU / mu according to the number of viable bacteria of Bacillus circulans DG-7. As another embodiment, the dose of the first leaf surface spraying is 4×10 10 ~4×10 11 CFU / mu according to the number of viable bacteria of Bacillus circulans DG-7. As an embodiment, the bacterial agent of the above technical solution is diluted 10-100 times by water, and the leaf surface spraying is carried out at the dosage of 20-40 L / mu. As another embodiment, the bacterial agent of the above technical solution is diluted 100 times by water, and the leaf surface spraying is carried out at the dosage of 30 L / mu.

[0032] After the angelica seedling is obtained, the angelica seedling is soaked by the bacterial agent of the above technical solution, and the soaked angelica seedling is obtained. As an embodiment, the soaking time is 30-60 min. As another embodiment, the soaking time is 40-50 min.

[0033] After the soaked angelica seedling is obtained, the soaked angelica seedling is transplanted, and the second leaf surface spraying is carried out on the transplanted angelica seedling by the bacterial agent of the above technical solution. As an embodiment, the second leaf surface spraying is carried out for 2-4 times. As another embodiment, the second leaf surface spraying is carried out for 4 times. As an embodiment, the dose of the second leaf surface spraying is 4×10 10 ~8×10 12 CFU / mu according to the number of viable bacteria of Bacillus circulans DG-7. As another embodiment, the dose of the second leaf surface spraying is 4×10 10 ~4×10 12 CFU / mu according to the number of viable bacteria of Bacillus circulans DG-7. As another embodiment, the dose of the second leaf surface spraying is 4×10 10 ~4×10 11As an embodiment, the present application dilutes the bacterial agent in water by 10-100 times, and sprays the second time on the leaves according to the dosage of 20-40 L / mu. As another embodiment, the present application dilutes the bacterial agent in water by 100 times, and sprays the second time on the leaves according to the dosage of 30 L / mu. As an embodiment, the interval between the two adjacent second leaf spraying is 2 weeks.

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

[0035] The reagents and culture media used in the experiment of the present application are 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 are purchased from Beijing Hezi Sheng Gong Technology Co., Ltd. The total plant RNA extraction kit, reverse transcription kit, and SYBR Green fluorescent quantitative kit are purchased from Tian Gen Biological Technology Co., Ltd.

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

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

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

[0039] Modified Stephenson culture 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 value is adjusted to 8.2.

[0040] NBRIP solid culture 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 value is 7.0±0.2.

[0041] 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.

[0042] Glucose proteose peptone water medium: glucose 0.5 g / L, proteose peptone 0.5 g / L, potassium phosphate dibasic 0.2 g / L, pH value is adjusted to 7.2 to 7.4.

[0043] Proteose peptone ammonification medium: proteose peptone 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 is adjusted to 7.2

[0044] In order to further illustrate the present application, a strain of Bacillus sp. DG-7 and its application in inhibiting the flowering of Angelica sinensis and improving the disease resistance of Angelica sinensis are described in detail below in combination with examples, but they cannot be understood as limiting the protection scope of the present application.

[0045] Example 1

[0046] Isolation and identification of Bacillus sp. DG-7

[0047]

[0048] Example 2

[0049] Function detection of Bacillus sp. DG-7

[0050] 1. Phosphorus solubilizing property determination: 10 μL of Bacillus sp. DG-7 bacterial solution of Example 1 was inoculated on NBRIP solid medium and incubated in a 30°C incubator. The phosphorus solubilizing circle and its size on the medium were observed periodically within 7 days. The phosphorus solubilizing ability of the strain was determined according to the size of the phosphorus solubilizing circle. The results showed that Bacillus sp. DG-7 did not solubilize phosphorus.

[0051] 2. Citrate utilization: Bacillus sp. DG-7 of Example 1 was inoculated on the center of a Simon's citrate agar medium with a loop, and the inoculation was repeated 3 times to evenly distribute the colonies. The inoculated plate was inverted and incubated in a 30°C incubator for 24-48 h. The color change around the colonies on the plate was observed, and a blue color change was positive. The results showed that Bacillus sp. DG-7 could not utilize citrate.

[0052] 3. Plate antagonism experiment: The antagonistic activity of Bacillus sp. DG-7 of Example 1 was determined by a plate confrontation method. Specifically, the fungal cakes of Fusarium and Alternaria pathogenic strains were inoculated on the center of PDA plates, and Bacillus sp. DG-7 of Example 1 was cross-inoculated at a distance of 2 cm from the pathogenic strains. The plates were incubated at a constant temperature of 28°C for 7 days. The colony growth conditions of the pathogenic fungi and the antagonistic bacteria were observed, the size of the inhibition zone was measured with a ruler, and the corresponding inhibition rate was calculated. The inhibition rate % = (inhibition zone radius - antagonistic bacteria radius) / antagonistic bacteria radius x 100%. The results showed that Bacillus sp. DG-7 had no plate antagonism to Fusarium and Alternaria pathogenic strains.

[0053] 4. Nitration power determination: Bacillus sp. DG-7 of Example 1 was inoculated in LB medium and incubated at 30°C and 180 r / min for 2 days. The bacterial cells were collected by centrifugation, resuspended in sterile water to OD 600 1.0, and then transferred into sterilized modified Stephenson medium. The medium was incubated at 30°C and 180 r / min for 2 days. The OD 600 value was measured, and the content was detected according to the method described in the nitrite nitrogen and nitrate nitrogen kit. The nitration power was calculated according to the following formula.

[0054]

[0055] After the calculation of the nitration power, the calculation result was divided by the turbidity of the corresponding bacterial solution, and the nitration power of the unit turbidity bacterial solution was standardized. Each strain was repeated 3 times, and the average value was calculated. The results showed that the nitration power of the unit turbidity bacterial solution of Bacillus sp. DG-7 was 1.662 ± 0.35%, which had strong nitration ability.

[0056] 5. Relative content of siderophore determination: The Bacillus sp. DG-7 of Example 1 was inoculated in LB medium and centrifuged to obtain the fermentation supernatant of siderophore (denoted as SCS). The SCS and CAS were mixed in a volume ratio of 1:1, and the mixture was incubated in a constant temperature water bath at 37°C for 0.5 h in the dark. The OD 630 of the mixture was determined. The relative content of siderophore of the strain was calculated according to the following formula: relative content of siderophore of the strain = (Ar-As) / Ar x 100%, wherein Ar is the OD 630 of the reference (the mixture of LB medium and CAS in a volume ratio of 1:1), and As is the OD 630 of the sample (the mixture of SCS and CAS). The calculated result was divided by the turbidity of the corresponding bacterial solution, and the relative content of siderophore of the unit turbidity bacterial solution was standardized. The results showed that the relative content of siderophore of Bacillus sp. DG-7 was 0.592% ± 0.13%.

[0057] 6. ACC deaminase activity determination of the strain: Bacillus sp. DG-7 of Example 1 was inoculated in LB medium and cultured at 30°C and 180 r / min for 1 d, then centrifuged at 4°C and 8000 r / min for 10 min. The supernatant was discarded, and the bacterial body was washed twice with DF medium without (NH4)2SO4. After centrifugation, the bacterial body was resuspended in ADF medium and cultured at 30°C and 180 r / min for 1 d. Then, the bacterial body was collected by centrifugation at 4°C and 8000 r / min for 10 min, washed twice with 0.1 mol / L Tris-HCl buffer at pH 7.6, resuspended in 0.2 mL Tris-HCl buffer at pH 8.5 with the same concentration, added with a small amount of toluene, and then the cells were broken by ultrasonic. Then, 20 μL of 0.5 mol / L 1-aminocyclopropane-1-carboxylic acid (ACC) was added, mixed, and incubated at 30°C for 15 min. Then, 0.3 mL of dinitrophenylhydrazine was added, covered, and incubated at 30°C 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 determined. 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 sp. DG-7 was 0.468 ± 0.68 μmol / min.

[0058] 7. Enzyme activity determination of the strain: Bacillus sp. DG-7 of Example 1 was inoculated in 50 mL of sterilized LB medium, cultured at 30°C and 180 r / min for 2 d, filtered, and the bacterial body was resuspended in sterile water to a turbidity OD 6001. The bacterial culture to be tested and the enzyme extract were mixed at a volume ratio of 1:10. The cells were disrupted by sonication in an ice bath, centrifuged at 8000 r / min for 10 min at 4℃, and the supernatant was placed on ice for testing. The activities of peroxidase, cellulase, neutral xylanase, and laccase were measured by spectrophotometry according to the instructions of the kits, and repeated 3 times. The results were divided by the turbidity of the corresponding bacterial culture and normalized to the enzyme activity per unit turbidity. The results showed that Bacillus motility 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, with relatively strong laccase activity.

[0059] 8. Methyl red test: Using a sterile inoculating loop, *Bacillus molybdenum* DG-7 from Example 1 was inoculated into sterile glucose-peptone water medium. No medium was inoculated as a control. The medium was incubated at 30°C for 2–5 days. Five drops of methyl red reagent were added to the medium, and the color change was observed; a red color indicated a positive result. The results showed that *Bacillus molybdenum* DG-7 has a strong ability to break down glucose and produce acid.

[0060] 9. Ammoniation test: Inoculation with Bacillus kineticus DG-7 fermentation broth (OD) from Example 1 600 Add 1.5–1.8 g of the solution to peptone-ammoniated medium and inoculate with 6 dilutions (10⁻⁶ g / L). -2 10 -3 10 -4 10 -5 10 -6 10 -7 Three tubes were inoculated for each dilution, with one tube of culture medium inoculated with sterile water as a control. The cultures were incubated at 30°C. 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 hue was checked to determine if ammonia had been produced. The results showed that *Bacillus molybdenum* DG-7 has a strong ammonification ability, indicating its strong protein-degrading capacity.

[0061] 10. Catalase Test: Pick a colony of Bacillus motility DG-7 from Example 1, place it on a clean glass slide, and add an appropriate amount of 3% hydrogen peroxide solution. The presence of bubbles within 1 minute indicates a positive result; the amount of bubbles is used to preliminarily determine the activity level. The results show that Bacillus motility DG-7 has strong catalase activity.

[0062] 11. Self-poisoning degradation: the base medium is deglucosated M9 medium, 50 mg / L ferulic acid and 50 mg / L anthraquinone are added as the sole carbon source respectively. Bacillus sp. DG-7 in Example 1 is inoculated into the above-mentioned medium and M9 medium respectively. The inoculated medium is cultured at 30℃, 180 r / min for 3-5 days, the growth conditions in the two media are compared, and the OD value is measured. The results show that Bacillus sp. DG-7 can degrade ferulic acid, but cannot degrade anthraquinone. 600

[0063] Example 3

[0064] Inhibition of Angelica sinensis early sprouting by Bacillus sp. DG-7

[0065] 1. Preparation of treatment solution

[0066] Bacillus sp. DG-7 in Example 1 is inoculated into PDB medium and cultured at 28℃, 180 r / min for 2 days, then diluted with water to obtain a Bacillus sp. DG-7 treatment solution with an effective viable bacterial count of 2×10 6 ~ 2×10 7 CFU / mL; PDB medium without bacteria is diluted with the Bacillus sp. DG-7 treatment solution to obtain a control treatment solution.

[0067] 2. Angelica sinensis seeds were used as experimental materials, and the variety was Mingui No. 1. The test was carried out in the Chinese herbal medicine planting base of Tianzhu County Green Energy Agricultural Technology Co., Ltd. from April to October in 2023 and 2024. The soil type was millet calcareous soil, and the pH value was 8.5; the soil was prepared at the end of April, and organic fertilizer (organic matter ≥45%, total nutrient N+P2O5+K2O ≥4%) 1000 kg / mu was applied. A single factor completely randomized design was used for the test, with treatment group (T) and control group (CK) for a total of 2 treatments, 3 replicates per treatment, and plot area of 30 m 2 (4 m x 7.5 m).

[0068] Angelica sinensis was sown in mid-June 2023, and foliar spraying was carried out 4 times from August to September, once every 2 weeks, with equal spraying volume in different plots. Among them, the treatment group (T) sprayed 20 L of Bacillus sp. DG-7 treatment solution per mu of land each time; the control group (CK) sprayed 20 L of control treatment solution per mu of land each time. Seedlings were raised in early October 2023, and stored by conventional method.

[0069] ​In April 2024, the seedlings with the rhizome head diameter of 0.6-1 cm were selected as the Angelica sinensis seedlings (the Angelica sinensis seedlings with the rhizome head diameter of less than 0.6 cm have low emergence rate and slow growth in the early spring low-temperature and heavy drought areas such as Tianzhu, which has great influence on the production of commercial medicinal materials). The corresponding Angelica sinensis seedlings were soaked in the treatment liquid of the treatment group (T) and the control group (CK) for 30 min, and then transplanted after draining the surface water. After emergence, the Angelica sinensis (without film planting) was sprayed with the corresponding treatment liquid every 2 weeks, a total of 4 times, and the spraying amount of different plots was equal. The field management was carried out according to the conventional measures.

[0070] In late August 2024, the number of bolting and the number of root rot disease beads were counted, and the bolting inhibition rate and the disease inhibition rate were calculated according to the following formula, and the results are shown in Table 1.

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

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

[0073] Table 1 Effect of microbial agent on early bolting and root rot of Angelica sinensis

[0074] Treatment 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] As can be seen from Table 1, after the Angelica sinensis was treated with Bacillus thuringiensis DG-7, the bolting rate was 44.30%, and the early bolting inhibition rate was 50.19%. Bacillus thuringiensis DG-7 can significantly inhibit the early bolting of Angelica sinensis and reduce the incidence of root rot.

[0077] Example 4

[0078] Effect of Bacillus thuringiensis DG-7 on endogenous hormone level of Angelica sinensis seedlings

[0079] 1. Preparation of treatment liquid

[0080] Bacillus thuringiensis DG-7 of Example 1 was inoculated into PDB medium, and cultured at 28°C with 180 r / min shaking for 2 days. The medium was diluted with water to obtain a treatment liquid of Bacillus thuringiensis DG-7 with an effective viable bacterial count of 2x10 6 ~ 2x10 7 CFU / mL; and PDB medium without bacteria was diluted by the same multiple as the treatment liquid of Bacillus thuringiensis DG-7 to obtain a control treatment liquid.

[0081] 2. Angelica sinensis seeds were used as experimental materials, and the variety was Mingui No. 1. The experiment was conducted in the Chinese herbal medicine planting base of Tianzhu Green Energy Agricultural Technology Co., Ltd. from April to October in 2023 and 2024. The soil type was calcareous soil, and the pH value was 8.5. The soil was prepared at the end of April, and 1000 kg / mu of organic fertilizer (organic matter ≥45%, total nutrient N+P2O5+K2O ≥4%) was applied. Single factor completely randomized design was used for the experiment, and 2 treatments (treatment group (DG-7) and control group (CK)) were set up, each with 3 replicates, and the plot area was 30 m 2 (4 m x 7.5 m).

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

[0083] 3. Endogenous hormone detection: The contents of endogenous hormones in Angelica sinensis seedlings were determined by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS). The contents of free abscisic acid, bound abscisic acid (abscisic acid glucose ester) were detected, and the sum of the two was calculated as total abscisic acid. The contents of indole-3-acetonitrile, indole acetic acid-valine methyl ester, indole-3-acetic acid, indole-3-acetic acid methyl ester, and indole acetic acid-aspartic acid were detected, and the sum of the aforementioned auxins was calculated as total auxin. The contents of 2-methylthioisopentenyladenosine, isopentenyladenosine, N6-isopentenyladenine, dihydrozeatin-7-glycoside, trans-zeatin-9-glycoside-5'-monophosphate, N-6-isopentenyladenosine 5'-monophosphate, trans-zeatin-9-β-glucoside, dihydrozeatin-O-glycoside, 6-furo[3H]aminopurine, and zeatin riboside were detected, and the sum of the aforementioned cytokinins was calculated as total cytokinin. The contents of gibberellin 3, gibberellin 3 precursor 5, gibberellin 1, gibberellin 1 precursor 20, gibberellin 7, gibberellin 4, and gibberellin 4, 7 precursor 9 were detected, and the sum of the aforementioned gibberellins was calculated as total gibberellin. The sum of gibberellins 1, 3, 4, and 7 was calculated 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 as total jasmonic acid. The contents of salicylic acid-2-O-β-glucoside and salicylic acid methyl ester O-β-glucoside were detected, and the sum of the two was calculated as bound salicylic acid. The contents of free salicylic acid, free salicylic acid precursor o-hydroxycinnamic acid, ethylene precursor 1-aminocyclopropane carboxylic acid (ACC), and melatonin were detected. The fold change (FC, average value of the treatment group / average value of the control group) and log2(FC) values were 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 - - p-Coumaric 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: In the table, lowercase letters indicate P < 0.05.

[0087] Table 2 shows that ACC and active gibberellin levels were zero, and melatonin levels were very low with no significant difference. Compared to the control, the DG-7 treatment group showed significant differences in the levels of other hormones: DG-7 treatment significantly reduced free abscisic acid (FA) levels (log2(FC) = -1.255) and significantly increased inactive bound AFA levels (log2(FC) = 1.334), resulting in a decrease in total AFA levels; a decrease in total auxin levels; an increase in total cytokinin levels; a decrease in total jasmonic acid levels; and an increase in free salicylic acid and its precursor o-hydroxycinnamic acid, while a decrease in bound salicylic acid levels, albeit with minor changes. In conclusion, Bacillus agglutinationis treatment with DG-7 significantly reduced AFA and auxin levels and increased free salicylic acid levels, thus inhibiting seedling growth, reducing root branching, improving resistance to biological stress, helping to reduce bolting rate, and enhancing disease resistance.

[0088] Example 5

[0089] Effects of Bacillus molybdenum DG-7 on the endogenous physiological and biochemical status and bolting-related gene expression in transplanted Angelica sinensis

[0090] 1. Preparation of the treatment solution

[0091] Bacillus kinosus DG-7 from Example 1 was inoculated into PDB medium and cultured at 28°C with shaking at 180 rpm for 2 days. The medium was then diluted with water to a viable count of 2 × 10⁻⁶. 6 ~2×10 7 CFU / mL was used to obtain the Bacillus motility DG-7 treatment solution; PDB medium without bacteria was diluted by the same factor as the Bacillus motility DG-7 treatment solution to obtain the control treatment solution.

[0092] 2. Using one-year-old Angelica sinensis seedlings (0.6–1 cm in diameter at the rhizome) corresponding to Example 4 as experimental material, the experiment was conducted from April to October 2024 at the medicinal herb planting base of Tianzhu County Lvneng Agricultural Technology Co., Ltd. The soil type was chestnut-calcareous soil with a pH of 8.5; land preparation was completed at the end of April, and 1000 kg / mu of organic fertilizer (organic matter ≥45%, total nutrients N+P2O5+K2O ≥4%) was applied. A single-factor completely randomized design was used for the experiment, with two treatments: a treatment group (DG-7) and a control group (CK), with three replicates per treatment. The plot area was 30 m². 2 (4m×7.5m).

[0093] The 1-year-old Angelica sinensis seedlings of the treatment group (DG-7) were soaked in the Bacillus thuringiensis DG-7 treatment solution for 30 min before transplanting; the 1-year-old Angelica sinensis seedlings of the control group (CK) were soaked in the control treatment solution for 30 min before transplanting; the surface water was drained before transplanting; after germination, the Angelica sinensis seedlings of the treatment group (DG-7) were sprayed with the Bacillus thuringiensis DG-7 treatment solution every 2 weeks, a total of 4 times, and the spraying amount was 20 L per mu each time; the Angelica sinensis seedlings of the control group (CK) were sprayed with the control treatment solution every 2 weeks, a total of 4 times, and the spraying amount was 20 L per mu each time. Three days after the fourth treatment, 10 Angelica sinensis seedlings were randomly selected from each plot, the third to fourth functional leaves at the same position were cut off, and the samples were mixed and stored in liquid nitrogen for later use.

[0094] 3. Physiological and biochemical index determination: according to the kit instructions, the activities of glutamine synthetase, glutamate synthetase, nitrite reductase, nitrate reductase, phenylalanine ammonia lyase, and the contents of ABTS, DPPH, total phenol, malondialdehyde, ammonium nitrogen, nitrate nitrogen, amino nitrogen, soluble sugar, chlorophyll, and flavonoids in the leaves of Angelica sinensis were determined by spectrophotometry. Each treatment was repeated 3 times, and the results are shown in Table 3.

[0095] Table 3. Results of physiological and biochemical index detection of Angelica sinensis during cultivation (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 Flavonoids (mg / g) 0.404±0.014b 0.530±0.005a 0.763 -0.389

[0097] Note: In the table, lowercase letters indicate P < 0.05.

[0098] According to Table 3, Bacillus thuringiensis DG-7 treatment had no significant effect on the removal of reactive oxygen species, significantly increased the contents of glutamine synthetase and glutamate synthetase, and reduced the contents of amino nitrogen and total nitrogen, indicating that Bacillus thuringiensis DG-7 had a certain inhibitory effect on the growth of Angelica sinensis; after Bacillus thuringiensis DG-7 treatment, the activity of PAL decreased, and the contents of total phenol and flavonoids decreased, indicating that Bacillus thuringiensis DG-7 treatment had the effect of reducing the content of total phenolic compounds.

[0099] 4. Endogenous hormone detection: the contents of endogenous hormones in the leaves of Angelica sinensis were determined by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS), and the determination indexes were the same as in Example 4. The results are shown in Table 4.

[0100] Table 4. Contents of endogenous hormones in Angelica sinensis during cultivation (x ± se, n = 3)

[0101]

[0102]

[0103] Note: In the table, lowercase letters indicate P < 0.05.

[0104] According to Table 4, except for the combination state of abscisic acid, there are significant differences in other indicators of DG-7 and CK treatment groups. Compared with the CK treatment group, the melatonin content of the strain DG-7 treatment was significantly increased and the degree was larger, with a log2(FC) value of 1.975; the combination state of salicylic acid content was significantly increased and the degree was larger, with a log2(FC) value of 3.204. The total abscisic acid and free abscisic acid contents were significantly decreased, which was consistent with the performance in the seedling stage; the auxin content was decreased, and the cytokinin content was increased, which was consistent with the performance in the seedling stage; the ACC content was increased; the active gibberellin and gibberellin total content was significantly higher than that in the seedling stage, and the inoculant treatment was significantly decreased; the jasmonic acid total was significantly decreased, which was consistent with the performance in the seedling stage; the free salicylic acid and its precursor and the combination state of salicylic acid content were significantly increased. In summary, the Bacillus sp. DG-7 treatment mainly increased the endogenous melatonin and salicylic acid contents, and reduced the abscisic acid, auxin and gibberellin contents, thereby effectively reducing the bolting rate.

[0105] 5. Detection of expression amount of bolting related genes

[0106] (1) RNA extraction: The angelica sample was quickly ground into powder in liquid nitrogen, and then a appropriate amount of lysis buffer was added and vortexed immediately. The mixture was transferred to a filter column CS and centrifuged at 12000 r / min for 2 min. The supernatant was collected and 0.5 times the volume of anhydrous ethanol was added. After mixing, the mixture was transferred to an adsorption column CR3. The subsequent operation was carried out according to the instructions. 50 μL of RNase-Free ddH2O was used for elution to obtain RNA, which was stored at -80℃.

[0107] (2) Reverse transcription: Reaction system (20 μL): 5×FastKing-RT SuperMix 4 μL, RNA 1 μg, RNase-Free ddH2O to 20 μL; Reaction program: 42℃, 15 min (remove genomic and reverse transcription reaction), 95℃, 3 min (enzyme inactivation process), obtain cDNA template.

[0108] (3) qRT-PC reference and different pathway key genes related to angelica bolting (Li Jie. Regulation mechanism of angelica bolting and flowering in photoperiod stage[D]. Gansu Agricultural University, 2021. DOI:10.27025 / d.cnki.ggsnu.2021.000082.), the primers were synthesized by Sheng Wu Bioengineering (Shanghai) Co., Ltd. The detailed sequences are shown in Table 5. Each sample was repeated 3 times, and the average value was taken. The relative expression amount results are shown in Table 6.

[0109] Table 5 qRT-PCR primer sequences

[0110]

[0111]

[0112] Table 6 Gene expression detection results (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] According to Table 6, the absolute value of log2(FC) greater than 1 is GAI, GA20OX1, GA2OX6, GA2OX8, AP1, SOC1, HD3A, CO3 and FD. GAI is a negative regulation gene of gibberellin synthesis, and is significantly up-regulated after the treatment of the microbial agent. GA20OX1, GA2OX6 and GA2OX8 are genes encoding gibberellin synthesis, and their expression is significantly down-regulated, which is consistent with the down-regulation of the physicochemical index gibberellin content. The photoperiod pathway genes HD3A, CO3 and FD and the integrated gene SOC1 are all key genes of positive regulation related to bolting, and their expression is significantly down-regulated after the treatment of the microbial agent, indicating that the treatment of Bacillus movens DG-7 can affect the photoperiod and play a role in inhibiting the early bolting of Angelica sinensis.

[0116] According to the above content, it can be seen that the Bacillus movens DG-7 provided by the present application can inhibit the early bolting of Angelica sinensis, reduce the bolting rate, improve the disease resistance of Angelica sinensis, and especially reduce the incidence of root rot of Angelica sinensis.

[0117] 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, but not all the embodiments, and people can also obtain other embodiments according to the present embodiment without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. A strain of Bacillus motility ( Bacillus mobilis DG-7, accession number CGMCCNo.31089.

2. An inoculant characterized in that, The bacterial agent comprises the Bacillus mobilis DG-7 of claim 1 and / or the fermentation liquor of the Bacillus mobilis DG-7 of claim 1.

3. The bacterial agent of claim 2, wherein The concentration of Bacillus agri DG-7 in the bacterial agent is 2 x 10 6 CFU / mL. The concentration of Bacillus agri DG-7 in the bacterial agent is 2 x 10 9 CFU / mL.

4. The use of the Bacillus mobilis DG-7 of claim 1 or the bacterial agent of claim 2 or 3 in inhibiting the flowering of Angelica sinensis and / or improving the disease resistance of Angelica sinensis. The improvement of the disease resistance of Angelica sinensis is the improvement of the resistance of Angelica sinensis to root rot.

5. A method for inhibiting bolting and / or increasing disease resistance in Angelica archangelica, characterized in that, The method comprises the following steps: After the Angelica sinensis is planted for 2-3 months, the seedlings are sprayed with the bacterial agent of claim 2 or 3 for the first time as the Angelica sinensis seedlings; The Angelica sinensis seedlings are soaked in the bacterial agent of claim 2 or 3 before being transplanted, and the Angelica sinensis seedlings after being transplanted are sprayed with the bacterial agent of claim 2 or 3 for the second time; The improvement of the disease resistance of Angelica sinensis is the improvement of the resistance of Angelica sinensis to root rot.

6. The method of claim 5, wherein, The soaking time is 30-60 minutes.

7. The method of claim 5, wherein, The first leaf surface spraying is 2-4 times, and the dosage of each spraying is 4×10 10 ~8×10 12 CFU / acre in terms of the viable bacterial count of Bacillus subtilis DG-7.

8. The method of claim 5, wherein, The second leaf surface spraying is 2-4 times, and the dosage of each spraying is 4×10 10 ~8×10 12 CFU / acre in terms of the viable cell number of Bacillus subtilis DG-7.

9. The method according to any one of claims 5 to 8, characterized in that, The interval between the first spraying for two adjacent times is 2 weeks; The interval between the second spraying for two adjacent times is 2 weeks.

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