A strain of Bacillus siamensis DG-39, microbial agent and its application in inhibiting early bolting of Angelica sinensis

Abstract

The present invention belongs to the technical field of biological agents, and specifically relates to a Bacillus siamensis DG-39, a bacterial agent thereof, and their application in inhibiting the early bolting of Angelica sinensis. The Bacillus siamensis DG-39 and the bacterial agent containing Bacillus siamensis DG-39 of the present invention mainly inhibit the early bolting of Angelica sinensis by increasing the endogenous melatonin content of Angelica sinensis, decreasing the contents of abscisic acid, auxin and gibberellin, promoting the circulation and utilization of nitrogen, effectively changing the physiological and biochemical state of Angelica sinensis, and down-regulating the expression of photoperiod-related genes. At the same time, the Bacillus siamensis DG-39 bacterial agent of the present invention has the characteristics of nitrogen fixation, non-phosphate solubilization and siderophore production, and can promote the absorption and utilization of nitrogen by plants to avoid the promoting effect of excessive phosphorus on bolting; meanwhile, Bacillus siamensis DG-39 has strong laccase activity and can remove phenolic amine substances that promote bolting, which helps to reduce the bolting rate.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological agents, and particularly relates to a Bacillus siamensis DG-39, a bacterial agent and its application in inhibiting the early bolting of Angelica sinensis. Background Art

[0002] Angelica sinensis (Oliv.) Diels is a perennial herb of the genus Angelica in the family Apiaceae. Angelica sinensis is native to alpine regions and is a low-temperature and long-day plant. In its individual development, it needs to go through two developmental stages from vegetative growth to reproductive growth: the vernalization stage (requiring a low temperature of 0-5°C) and the light stage (requiring a long day of more than 12 hours). Therefore, preventing and controlling early bolting is an important technical measure to improve the yield and quality of Angelica sinensis.

[0003] The early bolting of Angelica sinensis is affected by various factors. For example, if the seeds are too plump, or the seeds from plants that bolted early are used for seedling raising, they are likely to bolt early after transplantation; if the seedling raising is too early, the seedling age will be too long, the seedling roots will be too large and heavy, and excessive nutrition is likely to develop into early bolting plants; the size of the seedlings is related to early bolting. Large seedlings, branched seedlings, old seedlings and seedlings overwintering in the field, and seedlings dug too late have all met the conditions of the vernalization stage and winter low temperature and are likely to bolt early; the cultivation conditions also affect bolting. Adverse external conditions such as drought, poor soil, lack of fertilizer, low mountains, shady slopes, flat land planting, early planting, and serious pests and diseases are likely to cause the plants of Angelica sinensis to accelerate through the two stages of development and bolt early. Because in the individual development process of Angelica sinensis, there are obvious stages. When it transfers from vegetative growth to reproductive growth, that is, bolts and flowers, it must go through the vernalization stage and the light stage.

[0004] Although in the prior art, pesticides and plant growth regulators are also used to deal with the problem of increased bolting rate caused by cultivation conditions or extreme environments, and the bolting rate of Angelica sinensis can be effectively reduced, but at the same time, it has a greater impact on the quality of Angelica sinensis.

[0005] Bacillus siamensis has the functions of promoting plant growth and biological control of plant diseases, and has control effects on various plant diseases such as bacterial, fungal and nematode diseases. There are many Bacillus siamensis with disease resistance functions in the prior art. For example, Chinese Patent CN117229950A reported a strain of Bacillus siamensis CAU-Y3, which has strong antagonistic effects on Ralstonia solanacearum and Phytophthora parasitica var. nicotianae, and has obvious growth-promoting effects on maize seedlings; Chinese Patent CN116790422A reported a strain of Bacillus siamensis KD50, and its control effects on Marssonina coronaria, Sclerotinia sclerotiorum and Pythium spp. can reach more than 80%; Chinese Patent CN116083328A reported a strain of Bacillus siamensis ZXKN01, which can effectively control the root-knot nematode disease of Chinese medicinal materials of the genus Panax, and also has certain control effects on soil-borne fungal diseases and bacterial diseases. However, there is no relevant report on Bacillus siamensis with the function of inhibiting the early bolting of Angelica sinensis in the prior art. Summary of the Invention

[0006] The purpose of the present invention is to provide a strain of Bacillus siamensis DG-39, a bacterial agent thereof and their application in inhibiting the early bolting of Angelica sinensis. The Bacillus siamensis DG-39 and the bacterial agent containing the Bacillus siamensis DG-39 can inhibit the early bolting of Angelica sinensis, and the effect is remarkable.

[0007] The present invention provides a strain of Bacillus siamensis DG-39, and the preservation number of the Bacillus siamensis DG-39 is CGMCC No. 28908.

[0008] The present invention also provides a bacterial agent containing the Bacillus siamensis DG-39 described in the above technical solution.

[0009] Preferably, the active ingredient of the bacterial agent includes the cells of Bacillus siamensis DG-39 and / or the fermentation broth of Bacillus siamensis DG-39.

[0010] Preferably, the effective viable bacteria concentration of Bacillus siamensis DG-39 in the bacterial agent is 2×(10 6 ~10 7 ) CFU / mL.

[0011] The present invention also provides the application of the Bacillus siamensis DG-39 described in the above technical solution or the bacterial agent described in the above technical solution in one or more of the following five items:

[0012] 1) Inhibiting the early bolting of Angelica sinensis;

[0013] 2) Increasing the melatonin content of Angelica sinensis;

[0014] 3) Reduce the content of one or more hormones in Angelica sinensis, such as abscisic acid, auxin, and gibberellin;

[0015] 4) Promote the circulation and / or utilization of nitrogen in Angelica sinensis;

[0016] 5) Down-regulate the expression of genes related to photoperiod in Angelica sinensis.

[0017] Preferably, the genes related to photoperiod include one or more of Headingdate 3A, Constans3, FloweringlocusD, and Suppressorofoverexpression ofCO1 genes.

[0018] The present invention also provides a method for inhibiting the early bolting of Angelica sinensis, which includes the following steps: first, spray the foliar surface of Angelica sinensis seedlings with the bacterial agent described in the above technical solution; then soak the Angelica sinensis seedlings with the bacterial agent described in the above technical solution and transplant them, and then spray the foliar surface of Angelica sinensis seedlings during the cultivation period with the bacterial agent described in the above technical solution.

[0019] Preferably, when spraying the foliar surface of the Angelica sinensis seedlings, calculated by the viable bacteria count of Bacillus siamensis DG-39, the spraying amount each time is 4×10 10 ~8×10 11 CFU / mu, spray once every 2 weeks, and spray 2 to 4 times in total.

[0020] Preferably, when soaking the Angelica sinensis seedlings, the effective viable bacteria concentration of the bacterial agent is 2×10 6 ~2×10 7 CFU / mL, and the soaking time is 30 to 60 minutes.

[0021] Preferably, when spraying the foliar surface of the Angelica sinensis seedlings during the cultivation period, calculated by the viable bacteria count of Bacillus siamensis DG-39, the spraying amount each time is 4×10 10 ~8×10 11 CFU / mu, spray once every 2 weeks, and spray 2 to 4 times in total.

[0022] Beneficial effects:

[0023] The present invention provides a strain of Bacillus siamensis DG-39, which has been deposited. The deposit number of Bacillus siamensis DG-39 is CGMCC No. 28908.

[0024] Based on the Bacillus siamensis DG-39, the present invention also provides a bacterial agent containing Bacillus siamensis DG-39. The bacterial agent mainly increases the endogenous melatonin content of Angelica sinensis, reduces the contents of abscisic acid, auxin and gibberellin, and at the same time can promote the circulation and utilization of nitrogen, effectively change the physiological and biochemical state of Angelica sinensis, and play a role in inhibiting the early bolting of Angelica sinensis by down-regulating the expression of photoperiod-related genes.

[0025] At the same time, the Bacillus siamensis DG-39 and the bacterial agent containing Bacillus siamensis DG-39 of the present invention have the characteristics of nitrogen fixation, non-phosphate solubilization and siderophore production, can promote the absorption and utilization of nitrogen by plants, and avoid the promoting effect of excessive phosphorus on bolting; at the same time, Bacillus siamensis DG-39 has strong laccase activity and can remove phenolic amine substances that promote bolting, which helps to reduce the bolting rate.

[0026] Biological preservation information

[0027] Bacillus siamensis DG-39, with the biological classification of Bacillus siamensis, was deposited on November 08, 2023 at the China General Microbiological Culture Collection Center (CGMCC), with the deposit address being No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number being CGMCC No. 28908. Detailed implementation manners

[0028] The present invention provides a strain of Bacillus siamensis DG-39, and the deposit number of the Bacillus siamensis DG-39 is CGMCC No. 28908.

[0029] The Bacillus siamensis DG-39 of the present invention was isolated from a medicinal plant growing in Min County, Dingxi City, Gansu Province, China in alpine regions, and was identified as Bacillus siamensis by colony characteristics and 16S rDNA sequence. The colony characteristics of the Bacillus siamensis DG-39 of the present invention on LB solid medium are: the colony is round, light yellowish-white, with irregular edges, protruding in the middle, opaque, and the surface is wrinkled. The 16S rDNA sequence of the Bacillus siamensis DG-39 of the present invention is as shown in SEQ ID NO: 1, and has a similarity of more than 98.5% with the standard strain Bacillus siamensis KCTC 13613 strain.

[0030] The present invention also provides a bacterial agent containing the Bacillus siamensis DG-39 described in the above technical solution.

[0031] As an implementation manner, the active ingredient of the microbial agent of the present invention can be the cells of Bacillus siamensis DG-39 and / or the fermentation broth of Bacillus siamensis DG-39; as another implementation manner, the active ingredient of the microbial agent can be the cells of Bacillus siamensis DG-39 or the fermentation broth of Bacillus siamensis DG-39; as another implementation manner, the active ingredient of the microbial agent can be the fermentation broth of Bacillus siamensis DG-39. As an implementation manner, the effective viable bacteria concentration of Bacillus siamensis DG-39 in the microbial agent is 2×(10 6 ~10 7 ) CFU / mL.

[0032] As an implementation manner, the preparation method of the fermentation broth of Bacillus siamensis DG-39 of the present invention includes: inoculating Bacillus siamensis DG-39 into a liquid fermentation medium for fermentation culture to obtain the fermentation broth of Bacillus siamensis DG-39. As an implementation manner, the liquid fermentation medium of the present invention can be a PDB medium. As an implementation manner, the temperature of the fermentation culture can be 25-30 °C; as another implementation manner, the temperature of the fermentation culture can be 28 °C. As an implementation manner, the time of the fermentation culture can be 1-3 d; as another implementation manner, the time of the fermentation culture can be 2 d. As an implementation manner, the oscillation frequency of the fermentation culture can be 150-200 r / min; as another implementation manner, the oscillation frequency of the fermentation culture can be 180 r / min.

[0033] The microbial agent of Bacillus siamensis DG-39 of the present invention has the characteristics of nitrogen fixation, non-phosphate solubilization and siderophore production, and can promote the absorption and utilization of nitrogen by plants to avoid the promotion of bolting by excessive phosphorus; at the same time, Bacillus siamensis DG-39 has strong laccase activity and can remove phenolic amine substances that promote bolting, which helps to reduce the bolting rate. At the same time, the microbial agent of the present invention can increase the endogenous melatonin content of Angelica sinensis, reduce the contents of abscisic acid, auxin and gibberellin, and at the same time can promote the circulation and utilization of nitrogen, effectively change the physiological and biochemical state of Angelica sinensis, and play a role in inhibiting the early bolting of Angelica sinensis by down-regulating the expression of photoperiod-related genes.

[0034] Based on the above advantages, the present invention also provides the application of Bacillus siamensis DG-39 described in the above technical solution or the microbial agent described in the above technical solution in one or more of the following five items: 1) inhibiting the early bolting of Angelica sinensis; 2) increasing the melatonin content of Angelica sinensis; 3) reducing the content of one or more hormones among abscisic acid, auxin and gibberellin of Angelica sinensis; 4) promoting the circulation and / or utilization of nitrogen by Angelica sinensis; 5) down-regulating the expression of photoperiod-related genes of Angelica sinensis.

[0035] As an implementation manner, the photoperiod-related gene of the present invention may be one or more of Heading date 3A, Constans3, Flowering locusD, and Suppressor of overexpression of CO1 genes.

[0036] The present invention also provides a method for inhibiting the early bolting of Angelica sinensis, which includes the following steps: first, spraying the foliage of Angelica sinensis seedlings with the bacterial agent described in the above technical solution; then soaking the Angelica sinensis seedlings with the bacterial agent described in the above technical solution and transplanting them, and then spraying the foliage of the Angelica sinensis seedlings during the cultivation period with the bacterial agent described in the above technical solution.

[0037] As an implementation manner, when spraying the foliage of the Angelica sinensis seedlings of the present invention, based on the viable bacteria count of Bacillus siamensis DG-39, the spraying amount each time is 4×10 10 ~8×10 11 CFU / mu, spraying once every 2 weeks, for a total of 2 to 4 times; as another implementation manner, based on the viable bacteria count of Bacillus siamensis DG-39, the spraying amount each time is 4×10 10 ~4×10 11 CFU / mu, spraying once every 2 weeks, for a total of 4 times. As an implementation manner, the bacterial agent of the present invention may be the fermentation broth of Bacillus siamensis DG-39, and the characteristics of the fermentation broth of Bacillus siamensis DG-39 have been defined in the above technical solution and will not be elaborated here.

[0038] As an implementation manner, when soaking the Angelica sinensis seedlings, the effective viable bacteria concentration of the bacterial agent is 2×10 6 ~2×10 7 CFU / mL, and the soaking time is 30 to 60 min; as another implementation manner, the soaking time may be 30 to 45 min. As an implementation manner, when performing the soaking, the present invention selects Angelica sinensis seedlings with a diameter of 0.6 to 1 cm at the rhizome part for soaking.

[0039] As an implementation manner, when spraying the foliage of the Angelica sinensis seedlings during the cultivation period, based on the viable bacteria count of Bacillus siamensis DG-39, the spraying amount each time is 4×10 10 ~8×10 11 CFU / mu, spraying once every 2 weeks, for a total of 2 to 4 times. As another implementation manner, based on the viable bacteria count of Bacillus siamensis DG-39, the spraying amount each time is 4×10 10 ~4×10 11 CFU / mu, spraying once every 2 weeks, for a total of 4 times.

[0040] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0041] The test seeds and one-year-old Angelica sinensis seedlings (the diameter of the rhizome part is 0.6 - 1 cm) used in the examples are of the variety Min Gui No. 1, and were self-propagated by the inventor's research group in the Chinese herbal medicine planting base of Tianzhu Green Energy Agricultural Technology Co., Ltd. (altitude 2,600 meters). The experiments were carried out 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 is chestnut soil, and the pH value is 8.5; the land was prepared at the end of April, and 1,000 kg / mu of organic fertilizer (organic matter ≥ 45%, total nutrients N + P2O5 + K2O ≥ 4%) was applied.

[0042] The reagents and culture media used in the experiments are all chemically pure. Kits for detecting the activities of cellulase, laccase, xylanase, peroxidase, ACC deaminase, glutamine synthetase, glutamate synthase, nitrite reductase, nitrate reductase, phenylalanine ammonia-lyase, and kits for detecting the contents of ABTS, DPPH, siderophore, malondialdehyde, ammonium nitrogen, nitrate nitrogen, amino nitrogen, soluble sugar, chlorophyll, flavonoid, nitrite nitrogen, nitrate nitrogen were purchased from Beijing Boxbio Science & Technology Co., Ltd. The plant total RNA extraction kit, reverse transcription kit and SYBR Green fluorescence quantitative kit were purchased from Tiangen Biotech Co., Ltd.

[0043] The compositions of the culture media used in the following examples are as follows:

[0044] PDB medium: 200 g / L of potato, 20 g / L of glucose, natural pH.

[0045] LB medium: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, pH adjusted to 7.0 - 7.4.

[0046] 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, pH adjusted to 8.2.

[0047] 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, pH value of 7.0 ± 0.2.

[0048] Simmon's citrate agar medium: Sodium chloride 5.0 g / L, magnesium sulfate 0.2 g / L, ammonium dihydrogen phosphate 1.0 g / L, dipotassium hydrogen phosphate 1.0 g / L, sodium citrate 5.0 g / L, agar 20 g / L, 0.2% bromothymol blue solution 40 mL / L, adjust the pH to 6.8 ± 0.2.

[0049] Glucose peptone water medium: Glucose 0.5 g / L, peptone 0.5 g / L, dipotassium hydrogen phosphate 0.2 g / L, adjust the pH value to 7.2 - 7.4.

[0050] 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, adjust the pH value to 7.2.

[0051] DF medium formula: Basic components (per liter of distilled water): 1. Inorganic salt components: KH2PO4 4.0 g, Na2HPO4 6.0 g, MgSO4·7H2O 0.2 g, FeSO4·7H2O 0.1 g, CaCl2·2H2O 0.02 g, (NH4)2SO4 2.0 g; 2. Trace element solution (optional, add 1 mL per liter): H3BO3 0.3 g, CuSO4·5H2O 0.04 g, MnSO4·H2O 0.04 g, ZnSO4·7H2O 0.12 g, Na2MoO4·2H2O 0.025 g; 3. Carbon source: Glucose: 2.0 g (for initial culture to verify the growth ability of the strain).

[0052] ADF medium formula: Basic components (per liter of distilled water): 1. Inorganic salt components: KH2PO4 4.0 g, Na2HPO4 6.0 g, MgSO4·7H2O 0.2 g, FeSO4·7H2O 0.1 g, CaCl2·2H2O 0.02 g; 2. Trace element solution (optional, add 1 mL per liter): 2. Trace element solution (optional, add 1 mL per liter): H3BO3 0.3 g, CuSO4·5H2O 0.04 g, MnSO4·H2O 0.04 g, ZnSO4·7H2O 0.12 g, Na2MoO4·2H2O 0.025 g; 3. Carbon source: Glucose: 2.0 g (for initial culture to verify the growth ability of the strain); 4. Nitrogen source: ACC (1 - aminocyclopropane - 1 - carboxylic acid) 3.0 mM (for detecting ACC deaminase activity).

[0053] Example 1

[0054] Isolation and identification of Bacillus siamensis DG - 39, the steps are as follows:

[0055] A strain of bacteria isolated by the members of the inventor's research group from medicinal plants growing in Min County, Dingxi City, Gansu Province, China.

[0056] Min County is a high-cold area with an altitude range of 2040 - 3574 m and an average altitude of about 2500 m. It is located on the edge of the Qinghai-Tibet Plateau and is the transitional zone from Gannan Grassland to the Loess Plateau and the mountainous area of southern Gansu. The climate in Min County belongs to the transitional zone from temperate semi-humid to high-cold humid climate, with the characteristics of plateau continental climate, low temperature and dryness. The average annual temperature is about 6.1 °C, and the annual precipitation is about 600 mm. Due to its high altitude, the climate in Min County is complex and changeable, and extreme weather is easily formed.

[0057] The colony characteristics of the strain are as follows: The isolated strain was inoculated into LB solid medium and cultured at 30 °C for 2 days. The colony morphology of the strain was: the colony was round, light yellowish-white, with irregular edges, protruding in the middle, opaque, and the surface was wrinkled.

[0058]

[0059] Example 2

[0060] Functional detection of Bacillus siamensis DG-39 is carried out as follows:

[0061] 1) Determination of phosphorus-solubilizing characteristics: Take 10 μL of Bacillus siamensis DG-39 bacterial solution (obtained by culturing Bacillus siamensis DG-39 in LB medium at 28 °C and 180 r / min for 2 days) and inoculate it onto NBRIP solid medium, and place it in a constant temperature incubator at 30 °C. Regularly observe whether a phosphorus-solubilizing circle appears on the medium and its size within 7 days, and determine the phosphorus-solubilizing ability of the strain according to the size of the phosphorus-solubilizing circle of the strain.

[0062] The results show that Bacillus siamensis DG-39 does not solubilize phosphorus.

[0063] 2) Citrate utilization: Take an appropriate amount of Bacillus siamensis DG-39, and inoculate it in the center of Simmons citrate agar medium with an inoculation loop, and repeat the inoculation three times to make the colonies evenly distributed. Invert the inoculated plate and place it in a constant temperature incubator at 30 °C for 24-48 hours. Observe whether there is a color change around the colonies on the plate, and a change to blue is considered positive.

[0064] The results show that Bacillus siamensis DG-39 can utilize citrate.

[0065] 3) Nitrification determination: Inoculate Bacillus siamensis DG-39 into LB medium, shake culture 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, then inoculate it into the sterilized modified Stephenson medium, shake culture at 30 °C and 180 r / min for 2 days, measure the OD 600 value, detect the content according to the method described in the nitrite nitrogen and nitrate nitrogen kits, calculate the nitrification ability with the following formula, and divide the calculation result by the turbidity of the corresponding bacterial solution to standardize the nitrification ability of the bacterial solution with a unit turbidity. Repeat 3 times for each strain and calculate the average value.

[0066]

[0067] The results show that the nitrification ability of Bacillus siamensis DG-39 is 0.703 ± 0.05%.

[0068] 4) Determination of relative content of siderophore: Inoculate Bacillus siamensis DG-39 in LB medium, centrifuge after culturing to obtain the siderophore fermentation supernatant (SCS), mix the two solutions of SCS and chrome azurol S (CAS) at a volume ratio of 1:1, and measure the OD of the mixture after constant temperature water bath at 37 °C in the dark for 0.5 h 630, the relative content of siderophore of the strain is obtained by the following calculation formula. The relative content of siderophore of the strain = (Ar - As) / Ar × 100%, where Ar is the OD of the reference substance 630 (mixture of blank control and CAS); As is the OD of the sample 630 (mixture of strain SCS and CAS). The calculation result is divided by the turbidity of the corresponding bacterial solution, and standardized to the relative content of siderophore per unit turbidity of the bacterial solution.

[0069] The results show that the relative content of siderophore of Bacillus siamensis DG-39 is 0.607 ± 0.35%.

[0070] 5) Determination of ACC deaminase activity of the strain: Bacillus siamensis DG-39 was cultured in LB medium 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 (NH4)2SO4, 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, and the supernatant was discarded to collect the cells, which were washed twice with 0.1 mol / L Tris-HCl buffer at pH 7.6, centrifuged and resuspended in 0.2 mL of the same concentration Tris-HC1 buffer at pH 8.5. A small amount of toluene was added, and after ultrasonic cell disruption, 20 μL of 0.5 mol / L ACC was added. After mixing, it was incubated in a water bath at 30 °C for 15 min, then 0.3 mL of dinitrophenylhydrazine was added, the lid was covered, and it was reacted 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 is the content of α-ketobutyric acid produced per minute (μmol / min).

[0071] The results show that Bacillus siamensis DG-39 has no ACC deaminase activity.

[0072] 6) Determination of enzyme activity of the strain: Bacillus siamensis DG-39 was inoculated into 50 mL of sterilized LB medium and cultured at 30 °C and 180 r / min for 2 d, filtered, and the cells were collected and resuspended in sterile water to a turbidity OD 600 of 1 to obtain the bacterial solution to be tested. According to the volume ratio of the bacterial solution to be tested: enzyme extraction solution of 1:10, the cells were ultrasonically disrupted in an ice bath, centrifuged at 4 °C and 8000 r / min for 10 min, and the supernatant was placed on ice for testing. The spectrophotometric method described in the instruction manuals of peroxidase, cellulase, neutral xylanase, and laccase activity kits was used for determination, and repeated 3 times. The measurement result was divided by the turbidity of the corresponding bacterial solution, and standardized to the enzyme activity per unit turbidity of the bacterial solution.

[0073] The results showed that Bacillus siamensis DG-39 had no peroxidase and neutral xylanase activities, and its cellulase and laccase activities were 22.613 ± 0.245 and 2.848 ± 0.562 U / mL, respectively.

[0074] 7) Methyl red test: Inoculate Bacillus siamensis DG-39 into sterile glucose peptone water medium using a sterile inoculation loop. Use the uninoculated medium as a control. Incubate at 30 °C for 2 - 5 d. Add 5 drops of methyl red reagent to the medium and observe the color change. A red color indicates a positive result.

[0075] The results showed that Bacillus siamensis DG-39 had a strong ability to decompose glucose to produce acid.

[0076] 8) Ammonification test: Inoculate the fermentation broth of Bacillus siamensis DG-39 into peptone ammonification medium. Inoculate 6 dilutions (10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 ), with 3 tubes inoculated for each dilution. Take another tube of medium and inoculate it 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 broth onto a white porcelain colorimetric plate, add 2 drops of Nessler's reagent, and check if a brownish color appears to determine whether ammonia is produced.

[0077] The results showed that Bacillus siamensis DG-39 had a strong ammonification ability.

[0078] 9) Catalase test: Pick a colony of Bacillus siamensis DG-39 and place it on a clean glass slide. Add an appropriate amount of 3% hydrogen peroxide solution. If a large number of bubbles are produced within 1 min, it is a positive result.

[0079] The results showed that Bacillus siamensis DG-39 had no catalase activity.

[0080] 10) Degradation of autotoxic substances: Prepare a basal medium with inorganic salts and an appropriate nitrogen source, and add 50 mg / L of ferulic acid and anthraquinone as the sole carbon source respectively. Inoculate Bacillus siamensis DG-39 into the above medium. Incubate the inoculated medium at 30 °C and 180 r / min for 3 - 5 d, observe the color change of the medium and measure the OD 600 value.

[0081] The results showed that Bacillus siamensis DG-39 could degrade ferulic acid and anthraquinone.

[0082] Example 3

[0083] Bacillus siamensis DG-39 inhibits premature bolting of Angelica sinensis, and the steps are as follows:

[0084] Set up the experimental groups as follows:

[0085] Treatment group (T): The fermentation broth of Bacillus siamensis DG-39 cultured in PDB medium at 28 °C and 180 r / min for 2 days was diluted 100 times with water until the number of viable bacteria was 2×(10 6 ~10 7 ) CFU / mL. Spray 20 L per mu of land.

[0086] Positive control group (CK): PDB medium without bacteria, diluted by the same multiple as the treatment group, and spray 20 L per mu of land.

[0087] The experiment adopted a single-factor completely randomized design, with 2 treatments of T and CK, 3 replicates for each treatment, and the plot area was 30 m 2 (4 m×7.5 m). Sow in mid-June 2023, spray the leaves 4 times from August to September, once every 2 weeks. The spraying volume of different plots is equal. Lift the seedlings in early October and store them by conventional methods. In April 2024, before transplanting Angelica sinensis, select large seedlings with a diameter of 0.6 - 1 cm at the rhizome part, and soak the corresponding seedlings in the treatment group T and the positive control group CK for 30 min respectively, drain the surface moisture and transplant them. After emergence, spray the above treatments on the leaves of Angelica sinensis (without mulching), once every 2 weeks, for a total of 4 times. The spraying volume of different plots is equal. Field management is carried out according to conventional measures.

[0088] In late August 2024, count the number of bolted plants, and calculate the premature bolting inhibition rate according to the following formula: Premature bolting inhibition rate = (control bolting rate - treatment bolting rate) / control bolting rate.

[0089] After treating Angelica sinensis with Bacillus siamensis DG-39, the premature bolting situation is shown in Table 1.

[0090] Table 1 Effects of the microbial agent on premature bolting of Angelica sinensis

[0091] Experimental group Bolting rate / % Early bolting inhibition rate / % T 41.80±1.26b 52.91 CK 88.94±1.72a -

[0092] Note: Different lowercase letters in Table 1 indicate that compared with the CK group, the difference significance of the T group is P < 0.05.

[0093] Large seedlings of Angelica sinensis can better verify the bolting inhibition effect. Therefore, large seedlings with a diameter of 0.6 - 1 cm at the rhizome part are selected for the experiment. It can be seen from Table 1 that after treatment with Bacillus siamensis DG-39, the bolting rate of Angelica sinensis is 41.80%, and the premature bolting inhibition rate is 52.91% compared with CK.

[0094] Example 4

[0095] Effect of Bacillus siamensis DG-39 on the endogenous hormone levels of Angelica sinensis seedlings at the seedling stage, the steps are as follows:

[0096] Set up the experimental groups as follows:

[0097] Treatment group (T): The fermentation broth of Bacillus siamensis DG-39 cultured in PDB medium at 28 °C and 180 r / min for 2 days was diluted 100 times with water until the effective viable bacteria count was 2×(10 6 ~10 7 ) CFU / mL, and 20 L was sprayed per mu of land.

[0098] Positive control group (CK): PDB medium without bacteria, diluted by the same multiple as the treatment group, and 20 L was sprayed per mu of land.

[0099] The experiment adopted a single-factor completely randomized design, with 2 treatments of T and CK, and each treatment was repeated 3 times. Sowing was carried out in mid-June 2023, and each treatment was sprayed once every 2 weeks in early August, for a total of 4 times. In mid-September, 3 days after the 4th treatment, 30 Angelica sinensis seedlings were randomly selected from each plot, the leaves were picked, mixed samples were taken respectively, and stored in liquid nitrogen for later use.

[0100] Endogenous hormone detection:

[0101] The content of endogenous hormones in Angelica sinensis seedlings was determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS) (see Table 2). The detection indexes were: free abscisic acid, conjugated abscisic acid (abscisic acid glucosyl ester), total auxin (composed of indole-3-acetonitrile, indoleacetic acid-valine methyl ester, indole-3-acetic acid, indole-3-acetic acid methyl ester and indoleacetic acid-aspartic acid), total cytokinins (composed 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), the ethylene precursor 1-aminocyclopropane carboxylic acid (ACC), total gibberellins (composed of gibberellin 3, precursor 5 of gibberellin 3, gibberellin 1, precursor 20 of gibberellin 1, gibberellin 7, gibberellin 4 and precursor 9 of gibberellin 4,7), total jasmonic acid (composed of 12-hydroxyjasmonic acid, 12-oxo-phytodienoic acid, methyl jasmonate, jasmonic acid-isoleucine and jasmonic acid), melatonin, salicylic acid and precursors (composed of o-hydroxycinnamic acid and salicylic acid) and conjugated salicylic acid (composed of salicylic acid-2-O-β-glucoside and methyl salicylate O-β-glucoside).

[0102] Table 2 Endogenous hormones of Angelica sinensis seedlings (x±se, n = 3) ng / g

[0103] Index T CK FC log2(FC) Total abscisic acid 76.362±2.974b 182.399±1.957a 0.418 -1.256 Free abscisic acid 31.299±2.011b 157.738±2.825a 0.198 -2.333 Bound abscisic acid 45.063±2.968a 24.662±1.092b 1.827 0.869 Total auxin 9.039±0.023b 37.723±1.066a 0.239 -2.061 Total cytokinin 42.754±1.567a 32.765±1.072b 1.304 0.383 ACC 0 0 - - Total gibberellin 6.440±0.521 4.091±0.387 - - Total jasmonic acid 1270.792±18.821b 1804.186±20.274a 0.704 -0.505 Melatonin 0.559±0.022a 0.149±0.005b 3.639 1.863 Salicylic acid and precursors 230.045±1.483a 180.904±1.329b 1.271 0.346 Bound salicylic acid 299.003±5.819b 399.440±3.369a 0.748 -0.417

[0104] Note: Fold_Change: Fold difference (mean value of experimental group / mean value of control group); Log2FC: Taking the logarithm of the fold difference with base 2, the same below; Different lowercase letters in Table 2 indicate significant difference in the T group compared with the CK group, P < 0.05, the same below.

[0105] As can be seen from Table 2, except for ACC and gibberellin, there were significant differences in the contents of other hormones in the T group compared with the CK group. Specifically, the content of free abscisic acid in the T group decreased significantly, with a large decrease, and the value of log2(FC) was -2.333. The content of inactive bound abscisic acid increased, and the total content of abscisic acid decreased significantly, with the value of log2(FC) being -1.256. The total content of auxin decreased significantly, with the value of log2(FC) being -2.061. The content of melatonin increased significantly, with the value of log2(FC) being 1.863. Although salicylic acid and its precursors increased and bound salicylic acid decreased, the change degree was small. To sum up, the contents of gibberellin and melatonin in the seedling stage were very low. After treatment with Bacillus siamensis DG-39, the content of melatonin increased significantly, while the contents of abscisic acid and auxin decreased, which played a role in inhibiting seedling growth, reducing root branching, improving disease resistance, scavenging reactive oxygen species, and contributing to reducing the bolting rate.

[0106] Example 5

[0107] The steps for the effect of Bacillus siamensis DG-39 on the endogenous physiological and biochemical status and the expression of bolting-related genes of transplanted Angelica sinensis are as follows:

[0108] Treatment group (T): Dilute the fermentation broth of Bacillus siamensis DG-39 cultured in PDB medium at 28 °C and 180 r / min for 2 d with water by 100 times to an effective viable bacteria count of 2×(10 6 ~10 7 ) CFU / mL;

[0109] Positive control group (CK): PDB medium without bacteria, diluted by the same multiple as the treatment group;

[0110] Store the Angelica sinensis seedlings in winter and transplant them in mid-April. Soak them during transplantation. From May to June after transplanting and emergence, spray them once every 2 weeks for a total of 4 times. 3 days after the 4th treatment, randomly select 10 Angelica sinensis plants from 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.

[0111] (1) Determination of physiological and biochemical indexes: The equivalent quantitative values of ABTS scavenging ability, DPPH scavenging ability, total phenols, malondialdehyde, glutamine synthetase, glutamate synthetase, nitrite reductase, nitrate reductase, ammonium nitrogen, nitrate nitrogen, amino nitrogen, soluble sugar, chlorophyll, phenylalanine ammonia-lyase (PAL) activity and flavonoid content in Angelica sinensis leaves were determined by spectrophotometry as described in the kit. Each treatment was repeated 3 times, and the results are shown in Table 3.

[0112] Table 3 Detection of physiological and biochemical indexes during the cultivation period (x±se, n = 3)

[0113] Index T CK FC log2(FC) ABTS scavenging capacity equivalent quantification value (mmol / L) 0.146±0.002b 0.178±0.012a 0.830 -0.268 DPPH scavenging capacity equivalent quantification value (mmol / L) 0.475±0.023 0.506±0.014 - - Total phenols (mg / g) 1.222±0.021b 1.391±0.048a 0.877 -0.188 Malondialdehyde (nmol / g) 21.169±0.471a 14.612±0.352b 1.448 0.534 Glutamine synthetase (U / g) 8.318±0.291 8.319±0.109 - - Glutamate synthase (U / g) 96.807±0.618a 84.662±0.739b 1.143 0.193 Nitrite reductase (U / g) 2.585±0.088b 3.382±0.182a 0.763 -0.389 Nitrate reductase (U / g) 6.783±0.339a 2.689±0.691b 2.521 1.334 Ammonium nitrogen (μg / g) 69.685±0.236b 73.101±1.252a 0.953 -0.069 Nitrate nitrogen (μg / g) 17.455±2.541b 20.774±3.141a 0.840 -0.251 Amino nitrogen (μg / g) 166.955±3.317a 71.659±0.931b 2.329 1.220 Total nitrogen (μg / g) 256.116±0.827a 165.330±0.945b 1.549 0.631 Soluble sugar (mg / g) 25.442±0.671a 22.026±0.216b 1.155 0.208 Chlorophyll (mg / g) 6.177±0.017b 6.913±0.078a 0.893 -0.162 PAL (U / g) 113.545±3.435b 131.883±1.441a 0.861 -0.216 Flavonoids (mg / g) 0.401±0.042b 0.531±0.006a 0.754 -0.405

[0114] As can be seen from Table 3, there were no significant differences in the equivalent quantitative values of DPPH scavenging ability and glutamine synthetase, and significant differences existed in other indexes, but the degrees of difference were quite different. The indexes with the absolute value of log2(FC) greater than 1 were nitrate reductase and amino nitrogen. It indicated that the treatment with Bacillus siamensis DG-39 accelerated the nitrogen cycle, promoted the absorption and utilization of nitrogen by Angelica sinensis, and contributed to reducing the bolting rate.

[0115] (2) Detection of endogenous hormones: The content of endogenous hormones in Angelica sinensis leaves after 4 spraying treatments during the cultivation period was determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS). The detection indexes were the same as those in the seedling stage, and the results are shown in Table 4.

[0116] Table 4 Detection of endogenous hormones during the cultivation period (x±se, n = 3) ng / g

[0117]

[0118]

[0119] As can be seen from Table 4, significant differences existed in all indexes except conjugated abscisic acid. Compared with the CK group, the content of melatonin in the T group increased significantly and to a large extent, and the value of log2(FC) was 2.195; the content of conjugated salicylic acid increased significantly and to a large extent, and the value of log2(FC) was 2.337. In addition, the contents of total abscisic acid and free abscisic acid decreased significantly, which was consistent with the performance in the seedling stage; the content of auxin decreased, which was consistent with the performance in the seedling stage; the overall level of gibberellin content was significantly higher than that in the seedling stage, and the content decreased significantly after the treatment with the microbial agent. To sum up, the treatment with the microbial agent effectively reduced the bolting rate mainly by increasing the content of endogenous melatonin; decreasing the contents of abscisic acid, auxin and gibberellin; and promoting the nitrogen cycle and utilization.

[0120] (3) Detection of relative expression levels of bolting-related genes: 1) RNA extraction: The Angelica sinensis leaf samples collected after 4 foliar spraying treatments were quickly ground into powder in liquid nitrogen. After adding an appropriate amount of lysis buffer, vortex and mix immediately, transfer to the filter column CS, centrifuge at 12,000 r / min for 2 min, aspirate the supernatant and add anhydrous ethanol with a volume 0.5 times that of the supernatant. After mixing, transfer to the adsorption column CR3, and perform subsequent operations according to the instructions. Elute with 50 μL of RNase-free water to obtain RNA, and store at -80 °C. 2) Reverse transcription: Reaction system (20 μL): 4 μL of 5×FastKing-RT SuperMix, 1 μg of RNA, make up to 20 μL with RNase-free water; Reaction program: 42 °C, 15 min (to remove genomic DNA and perform reverse transcription reaction), 95 °C, 3 min (enzyme inactivation process) to obtain the cDNA template. 3) qRT-PCR: Refer to the key genes of different pathways related to Angelica sinensis bolting (see the literature

Li Jie. Research on the regulation mechanism of bolting and flowering of Angelica sinensis in the photoperiod stage [D]. Gansu Agricultural University, 2021.

[0121] Table 5 qRT-PCR primer sequences

[0122]

[0123]

[0124] Table 6 Detection of gene expression levels (x±se, n = 3)

[0125] Index T CK log2(FC) GAI 2.113±0.337a 1.022±0.249b 1.048 GA20OX1 0.724±0.134a 1.044±0.344a -0.527 GA2OX6 0.208±0.021b 1.003±0.103a -2.270 GA2OX8 0.943±0.067a 1.010±0.132a -0.099 AP1 0.015±0.002b 1.003±0.045a -6.004 SOC1 0.016±0.021b 1.021±0.049a -6.237 HD3A 0.188±0.022b 1.006±0.146a -2.419 MADS8 1.571±0.194a 1.004±0.120b 0.644 CO3 0.016±0.001b 1.027±0.277a -5.947 AGL8 0.555±0.092b 1.001±0.045a -0.851 FD 0.112±0.004b 1.010±0.132a -3.173

[0126] As can be seen from Table 6, compared with the CK group, although there were significant differences in the expression levels of different genes, the variation ranges of different indicators were quite different, and the greater the variation range, the greater the impact. The indicators with the absolute value of log2(FC) greater than 1 were GAI, GA2OX6, AP1, SOC1, HD3A, CO3, and FD. As a repressor of gibberellin biosynthesis, GAI inhibits gibberellin synthesis. GA2OX6 is a key gene for gibberellin synthesis. The up-regulation of GAI and the down-regulation of GA2OX6 reduced the endogenous gibberellin synthesis, which was consistent with the decrease in the gibberellin content of the physical and chemical indicators. The photoperiod pathway genes HD3A, CO3, FD and the integration gene SOC1 are all key positive regulatory genes for bolting, and their expression levels were down-regulated after the treatment with the microbial agent. It can be seen that the treatment with the microbial agent can also down-regulate the expression of photoperiod-related genes, playing a role in inhibiting the early bolting of Angelica sinensis.

[0127] It can be concluded from the above results that the Bacillus siamensis DG-39 of the present invention and the microbial agent containing the Bacillus siamensis DG-39 can inhibit the early bolting of Angelica sinensis, and the effect is remarkable.

[0128] 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 siamensis ( Bacillus siamensis ) DG-39, and the preservation number of the Bacillus siamensis DG-39 is CGMCC No. 28908.

2. A microbial agent containing the Bacillus siamensis DG-39 as described in claim 1.

3. The microbial agent according to claim 2, wherein The active ingredient of the microbial agent includes the cells of Bacillus siamensis DG-39 and / or the fermentation broth of Bacillus siamensis DG-39.

4. The microbial agent according to claim 2 or 3, characterized in that, The effective viable bacteria concentration of Bacillus siamensis DG-39 in the bacterial agent is 2×(10 6 ~10 7 ) CFU / mL.

5. Use of the Bacillus siamensis DG-39 as described in claim 1 or the microbial agent as described in any one of claims 2 to 4 in one or more of the following four aspects: 1) Inhibiting the early bolting of Angelica sinensis; 2) Increasing the melatonin content of Angelica sinensis; 3) Reducing the content of one or more hormones among abscisic acid, auxin and gibberellin in Angelica sinensis; 4) Promoting the utilization of nitrogen in Angelica sinensis.

6. A method for inhibiting the early bolting of Angelica sinensis, characterized in that, Comprising the following steps: first, spraying the foliage of Angelica sinensis seedlings with the microbial agent as described in any one of claims 2 to 4; then soaking the Angelica sinensis seedlings with the microbial agent as described in any one of claims 2 to 4 and transplanting them, and then spraying the foliage of Angelica sinensis seedlings during the cultivation period with the microbial agent as described in any one of claims 2 to 4.

7. The method according to claim 6, characterized in that When performing foliar spraying on the Angelica sinensis seedlings, based on the viable count of Bacillus siamensis DG-39, the spraying amount each time is 4×10 10 ~8×10 11 CFU / mu, and it is sprayed once every 2 weeks for a total of 2 to 4 times.

8. The method according to claim 6, wherein When soaking the Angelica sinensis seedlings, the effective viable bacteria concentration of the bacterial agent is 2×10 6 ~2×10 7 CFU / mL, and the soaking time is 30 - 60 min.

9. The method according to claim 6, wherein When performing foliar spraying on the Angelica sinensis seedlings during the cultivation period, based on the viable count of the Bacillus siamensis DG-39, the spraying amount each time is 4×10 10 ~8×10 11 CFU / mu, and it is sprayed once every 2 weeks for a total of 2 - 4 times.

Images