Bacillus siamensis DG-39, fungicide and application of bacillus siamensis DG-39 in inhibiting early bolting of angelica sinensis

Abstract

The invention belongs to the technical field of biological agents, and particularly relates to bacillus siamensis DG-39, a microbial agent and application of the bacillus siamensis DG-39 to inhibition of early bolting of angelica sinensis. According to the bacillus siamensis DG-39 and the microbial inoculum containing the bacillus siamensis DG-39, the physiological and biochemical states of angelica sinensis are effectively changed mainly by increasing the content of endogenous melatonin of angelica sinensis, reducing the content of abscisic acid, auxin and gibberellin and promoting circulation and utilization of nitrogen, and the photoperiod of the angelica sinensis can be effectively improved by reducing expression of related genes of the photoperiod. The effect of inhibiting early bolting of the angelica sinensis is achieved. Meanwhile, the bacillus siamensis DG-39 microbial inoculum has the characteristics of fixing nitrogen, not dissolving phosphorus and producing siderophore, and can promote the absorption and utilization of plants on nitrogen, so that the promotion effect of excessive phosphorus on bolting is avoided; meanwhile, the bacillus siamensis DG-39 laccase is high in activity, can remove phenolamine substances for promoting bolting, and is beneficial to reducing the bolting rate.

Description

Technical Field

[0001] The invention belongs to the technical field of biological microbial agents, and particularly relates to a siam bacillus DG-39, a microbial agent and application thereof in inhibiting early bolting of angelica sinensis. Background Art

[0002] Angelica sinensis (Oliv.) Diels is a perennial herbaceous plant of the Apiaceae family. Angelica sinensis is native to high-altitude mountainous areas and is a low-temperature, long-day plant. In individual development, it needs to go through two development stages from vegetative growth to reproductive growth: the vernalization stage (requiring a low temperature of 0 to 5°C) and the photoperiod stage (requiring a long day of more than 12 hours). Therefore, preventing and controlling premature bolting is an important technical measure to improve the yield and quality of Angelica sinensis.

[0003] The premature bolting of Angelica sinensis is affected by many factors, such as seeds that are too full, or seedlings with seeds from premature bolting plants are prone to premature bolting after transplanting; raising seedlings too early makes the seedlings too old, the roots too large, too heavy, and the nutrition too sufficient, which makes it easy for them to develop into premature bolting plants; the size of the seedlings is related to premature bolting. Large seedlings, forked seedlings, old seedlings, seedlings that overwinter in the field, and seedlings that are planted too late all have the conditions to accept the vernalization stage and low temperatures in winter, and are prone to premature bolting; cultivation conditions also affect bolting. Adverse external conditions such as drought, thin land, lack of fertilizer, low mountains, shady slopes, flat land planting, early planting, serious diseases and insect pests, etc., can easily cause Angelica sinensis plants to accelerate the transition between the two stages of development and bolt prematurely. This is because there are obvious stages in the individual development process of Angelica sinensis. When it switches from vegetative growth to reproductive growth, that is, bolting and flowering, it must go through the vernalization stage and the light stage.

[0004] Although the prior art also uses pesticides and plant growth regulators to deal with the problem of increased bolting rate caused by cultivation conditions or extreme environments, and can effectively reduce the bolting rate of angelica, reducing the bolting rate also has a significant impact on the quality of angelica.

[0005] Bacillus siamensis has plant growth promotion and disease biological control effects, and has control effects on plant diseases such as bacterial, fungal and nematode diseases. There are many Bacillus siamensis with disease resistance in the prior art. For example, Chinese patent CN117229950A reports a strain of Bacillus siamensis CAU-Y3, which has a strong antagonistic effect on tobacco solanacearum and tobacco black leg pathogen, and has an obvious growth-promoting effect on the growth of corn seedlings; Chinese patent CN116790422A reports a strain of Bacillus siamensis KD50, which has a control effect of more than 80% on apple disc Diplodia, Sclerotinia and Pythium; Chinese patent CN116083328A reports a strain of Bacillus siamensis ZXKN01, which can effectively control the root knot nematode disease of Panax genus Chinese medicinal materials, and also has a certain control effect on soil-borne fungal diseases and bacterial diseases. However, there is no relevant report on Bacillus siameensis that can inhibit the early bolting of Angelica sinensis in the prior art. Summary of the invention

[0006] The object of the present invention is to provide a strain of Siamese Bacillus DG-39, a bacterial agent and an application thereof in inhibiting the early bolting of Angelica sinensis. The Siamese Bacillus DG-39 and the bacterial agent containing the Siamese Bacillus DG-39 can inhibit the early bolting of Angelica sinensis with significant effect.

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

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

[0009] Preferably, the active ingredients of the bacterial agent include Bacillus siamensis DG-39 bacteria and / or fermentation broth of Bacillus siamensis DG-39.

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

[0011] The present invention also provides the use of the siam Bacillus 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) Inhibit the early bolting of Angelica sinensis;

[0013] 2) Increase the melatonin content of angelica sinensis;

[0014] 3) reducing the content of one or more hormones in angelica abscisic acid, auxin and gibberellin;

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

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

[0017] Preferably, the photoperiod-related genes include one or more of Headingdate 3A, Constans3, FloweringlocusD and Suppressor of overexpression of CO1 genes.

[0018] The present invention also provides a method for inhibiting early bolting of Angelica sinensis, comprising the following steps: first spraying the 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 then transplanting them, and then spraying the Angelica sinensis seedlings in the cultivation period with the bacterial agent described in the above technical solution.

[0019] Preferably, when the angelica seedlings are sprayed on the leaves, the spraying amount each time is 4×10 10 ~8×10 11 CFU / mu, spray once every 2 weeks, for a total of 2 to 4 times.

[0020] Preferably, when the angelica seedlings are soaked, the effective live bacteria concentration of the bacterial agent is 2×10 6 ~2×10 7 CFU / mL, the immersion time is 30 to 60 minutes.

[0021] Preferably, when the foliar spraying is performed on the angelica seedlings in the cultivation period, the spraying amount each time is 4×10 10 ~8×10 11 CFU / mu, spray once every 2 weeks, for a total of 2 to 4 times.

[0022] Beneficial effects:

[0023] The invention provides a siam bacillus DG-39, which has been biologically preserved. The preservation number of the siam bacillus DG-39 is CGMCC No.28908.

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

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

[0026] Biological deposit information

[0027] Bacillus siamensis DG-39, biologically classified as Bacillus siamensis, was deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration on November 8, 2023. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No. 28908. DETAILED DESCRIPTION

[0028] The 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 siam Bacillus DG-39 described in the present invention is isolated from a medicinal plant grown in Min County, Dingxi City, Gansu Province, China, a high-altitude cold region, and is confirmed to be siam Bacillus through colony characteristics and 16S rDNA sequence identification. The colony characteristics of the siam Bacillus DG-39 described in the present invention on LB solid culture medium are: the colony is round, light yellow-white, with irregular edges, protruding in the middle, opaque, and wrinkled on the surface. The 16SrDNA sequence of the siam Bacillus DG-39 described in the present invention is shown in SEQ ID NO: 1, and has a similarity of more than 98.5% with the standard strain Bacillus siamensis KCTC 13613.

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

[0031] As an embodiment, the active ingredient of the bacterial agent of the present invention may be the thallus of Bacillus siamensis DG-39 and / or the fermentation broth of Bacillus siamensis DG-39; as another embodiment, the active ingredient of the bacterial agent may be the thallus of Bacillus siamensis DG-39 or the fermentation broth of Bacillus siamensis DG-39; as another embodiment, the active ingredient of the bacterial agent may be the fermentation broth of Bacillus siamensis DG-39. As an embodiment, the effective live bacterial concentration of Bacillus siamensis DG-39 in the bacterial agent is 2×(10 6 ~10 7 )CFU / mL.

[0032] As an embodiment, the method for preparing the fermentation liquid of Bacillus siamese DG-39 of the present invention comprises: inoculating the Bacillus siamese DG-39 into a liquid fermentation medium for fermentation culture to obtain the fermentation liquid of Bacillus siamese DG-39. As an embodiment, the liquid fermentation medium of the present invention may be a PDB medium. As an embodiment, the temperature of the fermentation culture may be 25 to 30°C; as another embodiment, the temperature of the fermentation culture may be 28°C. As an embodiment, the fermentation culture time may be 1 to 3 days; as another embodiment, the fermentation culture time may be 2 days. As an embodiment, the oscillation frequency of the fermentation culture may be 150 to 200 r / min; as another embodiment, the oscillation frequency of the fermentation culture may be 180 r / min.

[0033] The bacterial agent of Siamese Bacillus DG-39 of the present invention has the characteristics of nitrogen fixation, non-phosphorus dissolution and siderophore production, and can promote the absorption and utilization of nitrogen by plants to avoid the promotion of excessive phosphorus on bolting; at the same time, Siamese Bacillus DG-39 has strong laccase activity, which can remove phenolamine substances that promote bolting, and help reduce the bolting rate. At the same time, the bacterial agent of the present invention can increase the endogenous melatonin content of Angelica sinensis, reduce the content of abscisic acid, auxin and gibberellin, and promote the circulation and utilization of nitrogen, effectively change the physiological and biochemical state of Angelica sinensis, and inhibit 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 use of the Siamese Bacillus DG-39 described in the above technical scheme or the bacterial agent described in the above technical scheme in one or more of the following five items: 1) inhibiting early bolting of Angelica sinensis; 2) increasing the melatonin content of Angelica sinensis; 3) reducing the content of one or more hormones in abscisic acid, auxin and gibberellin of Angelica sinensis; 4) promoting the circulation and / or utilization of nitrogen in Angelica sinensis; 5) down-regulating the expression of photoperiod-related genes of Angelica sinensis.

[0035] As an embodiment, 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 early bolting of Angelica sinensis, comprising the following steps: first spraying the 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 then transplanting them, and then spraying the Angelica sinensis seedlings in the cultivation period with the bacterial agent described in the above technical solution.

[0037] As an embodiment, when the angelica seedlings are sprayed on the leaves, the spraying amount each time is 4×10 10 ~8×10 11 CFU / mu, spray once every 2 weeks, for a total of 2 to 4 times; as another embodiment, the spraying amount each time is 4×10 10 ~4×10 11 CFU / mu, spray once every 2 weeks, a total of 4 times. As an embodiment, the bacterial agent of the present invention can be a fermentation broth of Siamese Bacillus DG-39, the characteristics of the fermentation broth of Siamese Bacillus DG-39 have been defined in the above technical solution and will not be repeated here.

[0038] As an embodiment, when the angelica seedlings are soaked, the effective live bacteria concentration of the bacterial agent is 2×10 6 ~2×10 7 CFU / mL, the soaking time is 30 to 60 minutes; as another embodiment, the soaking time can be 30 to 45 minutes. As an embodiment, when performing the soaking, the present invention selects Angelica sinensis seedlings with a diameter of 0.6 to 1 cm at the rhizome for soaking.

[0039] As an embodiment, when the angelica seedlings in the cultivation period are sprayed on the leaves, the spraying amount each time is 4×10 10 ~8×10 11 CFU / mu, spray once every 2 weeks, for a total of 2 to 4 times. As another embodiment, the spraying amount each time is 4×10 10 ~4×10 11 CFU / mu, spray once every 2 weeks, for a total of 4 times.

[0040] In order to further illustrate the present invention, the technical solutions provided by the present invention are 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 seedlings (diameter of the reed head is 0.6-1 cm) used in the embodiment are of Mingui No. 1 variety, which was bred by the inventor's research group at the Chinese medicinal material planting base of Tianzhu Green Energy Agricultural Technology Co., Ltd. (2600 meters above sea level). The experiment was carried out at the Chinese medicinal material planting base of Tianzhu Green Energy Agricultural Technology Co., Ltd. from April to October 2023 and 2024. The soil type is millet-calcium soil with a pH value of 8.5; the land was prepared at the end of April, and 1000 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 experiment were all chemically pure. Cellulase, laccase, xylanase, peroxidase, ACC deaminase, glutamine synthetase, glutamate synthase, nitrite reductase, nitrate reductase, phenylalanine ammonia lyase activity detection kits, and ABTS, DPPH, siderophore, malondialdehyde, ammonium nitrogen, nitrate nitrogen, amino nitrogen, soluble sugar, chlorophyll, flavonoids, nitrite nitrogen, nitrate nitrogen content detection kits were purchased from Beijing Box Biotechnology Co., Ltd., and plant total RNA extraction kit, reverse transcription kit and SYBR Green fluorescence quantification kit were purchased from Tiangen Biotechnology Co., Ltd.

[0043] The composition of the culture medium used in the following examples:

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

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

[0046] Modified Stephenson's medium: ammonium sulfate 2g / L, manganese sulfate 0.01g / L, sodium dihydrogen phosphate 0.25g / L, magnesium sulfate 0.03g / L, calcium carbonate 0.5g / L, potassium dihydrogen phosphate 0.75g / L, pH adjusted to 8.2.

[0047] 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 7.0±0.2.

[0048] Simon's citrate agar medium: sodium chloride 5.0 g / L, magnesium sulfate 0.2 g / L, ammonium dihydrogen phosphate 1.0 g / L, potassium dihydrogen 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 culture medium: glucose 0.5g / L, peptone 0.5g / L, dipotassium hydrogen phosphate 0.2g / L, pH value adjusted to 7.2-7.4.

[0050] Peptone ammoniation medium: peptone 5g / L, dipotassium hydrogen phosphate 0.5g / L, sodium chloride 0.25g / L, magnesium sulfate heptahydrate 0.5g / L, ferrous sulfate 0.01g / L, adjust the pH to 7.2.

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

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

[0053] Example 1

[0054] The isolation and identification of Bacillus siamensis DG-39 was carried out as follows:

[0055] A fungus isolated by the inventor's research team members from a medicinal plant grown in Min County, Dingxi City, Gansu Province, China.

[0056] Min County is a high-altitude area with an altitude range of 2040-3574m and an average altitude of about 2500m. It is located on the edge of the Qinghai-Tibet Plateau and is a transition zone from the Gannan grassland to the Loess Plateau and Longnan Mountains. Min County's climate belongs to the transition zone from temperate semi-humid to high-altitude humid climate, with plateau continental climate characteristics, low temperature and dryness, with an average annual temperature of about 6.1℃ and an annual precipitation of about 600 mm. Due to its location at a high altitude, Min County's climate is complex and changeable, and extreme weather is prone to occur.

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

[0058]

[0059] Example 2

[0060] Functional detection of Bacillus siamensis DG-39, the steps are as follows:

[0061] 1) Determination of phosphate solubilization characteristics: Take 10 μL of Siamese Bacillus DG-39 bacterial liquid (obtained by culturing Siamese Bacillus DG-39 in LB medium at 28°C and 180 r / min for 2 days) and inoculate it on NBRIP solid culture medium, place it in a constant temperature culture at 30°C, and regularly observe whether a phosphate solubilization circle is generated on the culture medium and its size within 7 days. The phosphate solubilization ability of the strain is determined based on the size of the phosphate solubilization circle.

[0062] The results showed that Bacillus siamensis DG-39 did not solubilize phosphate.

[0063] 2) Citrate utilization: Take an appropriate amount of Bacillus siameensis DG-39 and inoculate it in the center of Simon's citrate agar medium with an inoculation loop. Repeat the inoculation three times to make the colonies evenly distributed. Place the inoculated plate upside down in a 30℃ constant temperature incubator and incubate for 24 to 48 hours. Observe whether there is a color change around the colonies on the plate. If it turns blue, it is positive.

[0064] The results showed that Bacillus siamese DG-39 could utilize citrate.

[0065] 3) Nitrification capacity determination: Bacillus siameensis DG-39 was inoculated into LB medium and cultured at 30°C and 180 rpm for 2 days. The cells were collected by centrifugation and resuspended in sterile water to an OD of 600 The OD value was 1.0, and then the culture medium was sterilized and cultured at 30℃ and 180r / min for 2 days. 600 The value was determined by the method described in the nitrite nitrogen and nitrate nitrogen kits. The nitrification capacity was calculated using the following formula. The result was divided by the turbidity of the corresponding bacterial solution and standardized to the nitrification capacity of the bacterial solution per unit turbidity. Each strain was repeated 3 times and the average value was calculated.

[0066]

[0067] The results showed that the nitrification capacity of Bacillus siameensis DG-39 was 0.703±0.05%.

[0068] 4) Determination of relative content of siderophore: Bacillus siameensis DG-39 was inoculated into LB medium and centrifuged to obtain siderophore fermentation supernatant (SCS). SCS and CAS were mixed at 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 and the OD of the mixture was measured. 630The relative siderophore content of the strain is calculated by the following formula: Relative siderophore content 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 sample OD 630 (Mixture of strains SCS and CAS). The calculated result was divided by the turbidity of the corresponding bacterial solution and standardized to the relative content of siderophore per unit turbidity bacterial solution.

[0069] The results showed that the relative content of siderophore in Bacillus siamese DG-39 was 0.607±0.35%.

[0070] 5) Determination of ACC deaminase activity produced by the strain: Bacillus siamese DG-39 was cultured in LB medium at 30°C and 180 r / min with shaking for 1 day, and then centrifuged at 4°C and 8000 r / min for 10 min. The supernatant was discarded, and the cells were washed twice with DF medium without (NH4)2SO4. After centrifugation, the cells were resuspended in ADF medium and cultured at 30°C and 180 r / min for 1 day. After that, centrifuge at 4℃ and 8000r / min for 10min, discard the supernatant and collect the cells, wash twice with 0.1mol / LTris-HCl buffer with pH value 7.6, resuspend in 0.2mL Tris-HCl buffer with pH value 8.5, add a small amount of toluene, ultrasonically disrupt the cells, add 20μL 0.5mol / LACC, mix well, bathe at 30℃ for 15min, add 0.3mL dinitrophenylhydrazine, cover the lid, place in 30℃ warm water bath for 0.5h; then add 2mL, 2mol / LNaOH to terminate the reaction, and measure the absorbance at 540nm. The enzyme activity of ACC deaminase is the content of α-ketobutyric acid produced per minute (μmol / min).

[0071] The results showed that Bacillus siamese DG-39 had no ACC deaminase activity.

[0072] 6) Determination of enzyme activity of strains: Bacillus siameensis DG-39 was inoculated into 50 mL of sterilized LB medium, cultured at 30°C and 180 rpm for 2 days, filtered, collected, and resuspended in sterile water to a turbidity OD 600 =1, and obtain the test bacterial solution. According to the volume ratio of the test bacterial solution to the enzyme extract of 1:10, the cells were broken by ultrasonication in an ice bath, centrifuged at 4°C and 8000r / min for 10min, and the supernatant was placed on ice for testing. The peroxidase, cellulase, neutral xylanase, and laccase activity kits were measured by spectrophotometry as described in the instructions, and repeated 3 times. The measurement results were divided by the turbidity of the corresponding bacterial solution and standardized to the enzyme activity of the unit turbidity bacterial solution.

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

[0074] 7) Methyl red test: Use a sterile inoculation loop to inoculate Bacillus siameensis DG-39 into a sterile glucose-peptone water culture medium. Do not inoculate the culture medium as a control. Incubate at 30°C for 2 to 5 days. Add 5 drops of methyl red reagent to the culture medium and observe the color change. A red color is positive.

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

[0076] 8) Ammoniation test: Inoculate the fermentation broth of Bacillus siamese DG-39 into peptone ammoniation medium, and inoculate 6 dilutions (10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 ), inoculate 3 tubes for each dilution, and take another tube of culture medium to inoculate sterile water as a control. Culture in a 30℃ constant temperature incubator. Check the turbidity of the culture medium on the 3rd and 5th day after culture. On the 7th day of culture, draw 5 drops of culture solution onto a white porcelain colorimetric plate, add two drops of Ghana's reagent, and check whether brown appears to determine whether ammonia is produced.

[0077] The results showed that Bacillus siameensis DG-39 had strong ammoniation ability.

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

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

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

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

[0082] Example 3

[0083] Siamese Bacillus DG-39 inhibits the early sprouting of Angelica sinensis, the steps are as follows:

[0084] Set up the test group as follows:

[0085] Treatment group (T): The fermentation liquid 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 to a viable count of 2×(10 6 ~10 7 ) CFU / mL. Spray 20L per acre.

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

[0087] The experiment adopted a single-factor completely randomized design, with two treatments, T and CK, and three replicates per treatment. The plot area was 30m 2 (4m×7.5m). Sowing was carried out in mid-June 2023, and foliar spraying was performed 4 times from August to September, with spraying once every 2 weeks. The spraying amount and volume in different plots were equal. The seedlings were removed in early October and stored using conventional methods. In April 2024, before transplanting Angelica sinensis, large seedlings with a diameter of 0.6 to 1 cm at the reed head were selected. The corresponding seedlings were soaked in the treatment group T and the positive control group CK for 30 minutes, respectively, and the surface moisture was drained and transplanted. After emergence, the Angelica sinensis (planted without film) was sprayed with the above treatment on the leaves, with foliar spraying once every 2 weeks, for a total of 4 times, with spraying amount and volume in different plots being equal. Field management was carried out according to conventional measures.

[0088] At the end of August 2024, the number of boltings was counted, and the early bolting inhibition rate was calculated according to the following formula: early bolting inhibition rate = (control bolting rate - treatment bolting rate) / control bolting rate.

[0089] The early sprouting situation of Angelica sinensis after being treated with Bacillus siamense DG-39 is shown in Table 1.

[0090] Table 1 Effects of bacterial agents on early stems of Angelica sinensis

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

[0092] Note: Different lowercase letters in Table 1 indicate significant differences between group T and group CK (P < 0.05).

[0093] Large seedlings of Angelica sinensis can better verify the effect of inhibiting early bolting, so large seedlings with a diameter of 0.6 to 1 cm at the head of the rhizome were selected for the test. It can be concluded from Table 1 that after being treated with Siamese Bacillus DG-39, the bolting rate of Angelica sinensis was 41.80%, and compared with CK, the early bolting inhibition rate was 52.91%.

[0094] Example 4

[0095] The effect of Bacillus siameensis DG-39 on the endogenous hormone levels of Angelica sinensis seedlings was studied in the following steps:

[0096] Set up the test group as follows:

[0097] Treatment group (T): The fermentation liquid 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 to a count of 2×(10 6 ~10 7 )CFU / mL, spray 20L per acre.

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

[0099] The experiment adopted a single-factor completely randomized design, with two treatments, T and CK, and three replicates for each treatment. The seeds were sown in mid-June 2023, and sprayed once every two weeks in early August, for a total of four times. In mid-September, 3 days after the fourth treatment, 30 Angelica sinensis seedlings were randomly selected from each plot, leaves were picked, mixed, and frozen in liquid nitrogen for later use.

[0100] Endogenous hormone detection:

[0101] Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS) was used to determine the endogenous hormone content of Angelica sinensis seedlings (see Table 2). The detection indicators were: free abscisic acid, bound abscisic acid (abscisic acid glucose 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 cytokinin (composed of 2-methylthioisopentenyl adenosine nucleoside, isopentenyl adenosine nucleoside, N6-isopentenyl adenine, dihydrozeatin-7-glycoside, trans-zeatin-9-glycoside-5'-monophosphate, N-6-isopentenyl adenosine 5'-monophosphate, trans-zeatin-9-β-glucosidase glucoside, dihydrozemidine-O-glycoside, 6-furfurylaminopurine and zemidine riboside), 1-aminocyclopropanecarboxylic acid (ACC), the precursor of ethylene, the sum of gibberellins (consisting of gibberellin 3, the precursor of gibberellin 3 5, gibberellin 1, the precursor of gibberellin 1 20, gibberellin 7, gibberellin 4 and the precursor of gibberellin 4,7 9), the sum of jasmonic acids (consisting of 12-hydroxyjasmonic acid, 12-oxy-phyto-dienoic acid, methyl jasmonate, jasmonic acid-isoleucine and jasmonic acid), melatonin, salicylic acid and its precursor (consisting of o-hydroxycinnamic acid and salicylic acid) and bound salicylic acid (consisting of salicylic acid-2-O-β-glucoside and methyl salicylate O-β-glucoside).

[0102] Table 2 Endogenous hormones in 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 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 - - Gibberellins Sum 6.440±0.521 4.091±0.387 - - Sum of jasmonic acids 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 Conjugated salicylic acid 299.003±5.819b 399.440±3.369a 0.748 -0.417

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

[0105] As shown in 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 T group reduced the content of free abscisic acid to a greater extent, with a log2(FC) value of -2.333, increased the content of inactive bound abscisic acid, and reduced the content of total abscisic acid to a greater extent, with a log2(FC) value of -1.256; the total content of auxin decreased to a greater extent, with a log2(FC) value of -2.061; the content of melatonin increased to a greater extent, with a log2(FC) value of 1.863; although salicylic acid and its precursors increased and bound salicylic acid decreased, the degree of change was small. In summary, the content of gibberellin and melatonin in the seedling stage was very low. The treatment with Siamese Bacillus DG-39 greatly increased the content of melatonin and reduced the content of abscisic acid and auxin, which played a role in inhibiting seedling growth, reducing root branching, improving disease resistance, and removing reactive oxygen, which helped to reduce the bolting rate.

[0106] Example 5

[0107] The effects of Bacillus siameensis DG-39 on the endogenous physiological and biochemical states of transplanted Angelica sinensis and the expression of bolting-related genes were studied as follows:

[0108] Treatment group (T): The fermentation liquid 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 to a count of 2×(10 6 ~10 7 )CFU / mL;

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

[0110] Store the angelica seedlings in winter and transplant them in mid-April. Soak them during transplanting. Spray them once every 2 weeks in 5-6 months after the seedlings emerge, for a total of 4 times. 3 days after the 4th treatment, randomly select 10 angelica plants from each plot, cut off the 3rd and 4th functional leaves in the same position, mix them separately, and freeze them in liquid nitrogen for later use.

[0111] (1) Physiological and biochemical index determination: The ABTS scavenging capacity equivalent quantitative value, DPPH scavenging capacity equivalent quantitative value, total phenol, malondialdehyde, glutamine synthetase, glutamate synthetase, nitrite reductase, nitrate reductase, ammonium nitrogen, nitrate nitrogen, amino nitrogen, soluble sugar, chlorophyll, phenylalanine ammonia lyase (PAL) activity and flavonoid content of Angelica sinensis leaves were determined by spectrophotometry according to the kit. Each treatment was repeated 3 times, and the results are shown in Table 3.

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

[0113] index T CK FC log2(FC) ABTS clearance equivalent quantitative value (mmol / L) 0.146±0.002b 0.178±0.012a 0.830 -0.268 DPPH scavenging capacity equivalent quantitative 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 shown in Table 3, there were no significant differences in the equivalent quantitative value of DPPH scavenging capacity and glutamine synthetase, and there were significant differences in other indicators, but the degree of difference was relatively large. The indicators with absolute values ​​of log2(FC) greater than 1 were nitrate reductase and amino nitrogen. This showed that the treatment with Siamese Bacillus DG-39 accelerated the nitrogen cycle, promoted the absorption and utilization of nitrogen by Angelica sinensis, and helped to reduce the bolting rate.

[0115] (2) Endogenous hormone detection: Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS) was used to determine the endogenous hormone content in Angelica sinensis leaves after four spraying treatments during the cultivation period. The determination indicators were the same as those during the seedling stage. The results are shown in Table 4.

[0116] Table 4 Endogenous hormone detection during the cultivation period (x±se, n=3) ng / g

[0117]

[0118]

[0119] As shown in Table 4, except for bound abscisic acid, other indicators showed significant differences. Compared with the CK group, the melatonin content in the T group increased significantly and to a greater extent, with a log2(FC) value of 2.195; the bound salicylic acid content increased significantly and to a greater extent, with a log2(FC) value of 2.337. In addition, the total abscisic acid and free abscisic acid contents decreased significantly, which was consistent with the performance in the seedling stage; the auxin content decreased, 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 bacterial agent treatment significantly decreased. In summary, the bacterial agent treatment mainly increased the endogenous melatonin content; reduced the content of abscisic acid, auxin, and gibberellin; and promoted nitrogen circulation and utilization, thereby effectively reducing the bolting rate.

[0120] (3) Detection of relative expression 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, and immediately vortexed and mixed after adding an appropriate amount of lysis solution, and transferred to the filter column CS, centrifuged at 12000r / min for 2min, the supernatant was aspirated and 0.5 times the volume of anhydrous ethanol was added to the supernatant, and then transferred to the adsorption column CR3 after mixing. The subsequent operations were carried out according to the instructions, and RNA was eluted with 50μL of RNase-free water and stored at -80℃. 2) Reverse transcription: Reaction system (20μL): 5×FastKing-RT SuperMix 4μL, RNA 1μg, RNase-free water was added to 20μL; reaction procedure: 42℃, 15min (removal of genome and reverse transcription reaction), 95℃, 3min (enzyme inactivation process), to obtain cDNA template. 3) qRT-PCR: Referring to the key genes of different pathways related to Angelica sinensis bolting (see the literature [Li Jie. Study on the regulatory mechanism of Angelica sinensis bolting and flowering during the photoperiod stage [D]. Gansu Agricultural University, 2021.]), the primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the detailed sequences are shown in Table 5. Each sample was repeated 3 times, and the housekeeping gene Actin was used as the internal reference gene. The relative expression of genes was calculated by the method and the average value was taken. The results are shown in Table 6.

[0121] Table 5 qRT-PCR primer sequences

[0122]

[0123]

[0124] Table 6 Gene expression detection (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 shown in Table 6, compared with the CK group, although there are significant differences in the expression of different genes, the changes in different indicators are quite different, and the greater the change, the greater the impact. The indicators with an absolute value of log2(FC) greater than 1 are GAI, GA2OX6, AP1, SOC1, HD3A, CO3, and FD. GAI, as a repressor of gibberellin biosynthesis, inhibits gibberellin synthesis. GA2OX6 is a key gene for gibberellin synthesis. GAI is upregulated and GA2OX6 is downregulated, which reduces endogenous gibberellin synthesis, which is consistent with the decrease in the content of gibberellin in the physical and chemical indicators. The photoperiod pathway genes HD3A, CO3, FD and the integration gene SOC1 are all key genes for positive regulation of bolting, and their expression levels are downregulated by the treatment of bacterial agents. It can be seen that the treatment of bacterial agents can also downregulate the expression of photoperiod-related genes and play a role in inhibiting the early bolting of Angelica sinensis.

[0127] From the above results, it can be concluded that the Siamese Bacillus DG-39 and the bacterial agent containing the Siamese Bacillus DG-39 of the present invention can inhibit the early bolting of Angelica sinensis, and the effect is significant.

[0128] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus siamensis DG-39, the deposit number of which is CGMCC No.28908.

2. A bacterial agent containing the siam bacillus DG-39 according to claim 1.

3. The bacterial agent according to claim 2, characterized in that The active ingredients of the bacterial agent include siam Bacillus DG-39 bacteria and / or fermentation liquid of siam Bacillus DG-39.

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

5. Use of the siam bacillus DG-39 according to claim 1 or the bacterial agent according to any one of claims 2 to 4 in one or more of the following five items: 1) Inhibit the early bolting of Angelica sinensis; 2) Increase the melatonin content of angelica sinensis; 3) reducing the content of one or more hormones in angelica abscisic acid, auxin and gibberellin; 4) Promote the cycling and / or utilization of nitrogen in Angelica sinensis; 5) Down-regulate the expression of photoperiod-related genes in Angelica sinensis.

6. The use according to claim 5, characterized in that: The photoperiod-related genes include one or more of Heading date3A, Constans3, Flowering locus D and Suppressor of overexpression of CO1 genes.

7. A method for inhibiting early bolting of Angelica sinensis, characterized in that: The method comprises the following steps: first spraying the angelica seedlings with the microbial agent described in any one of claims 2 to 4; then immersing the angelica seedlings with the microbial agent described in any one of claims 2 to 4 and then transplanting them; and then spraying the angelica seedlings in the cultivation period with the microbial agent described in any one of claims 2 to 4.

8. The method according to claim 7, characterized in that When the angelica seedlings are sprayed on the leaves, the amount of each spray is 4×10 10 ~8×10 11 CFU / mu, spray once every 2 weeks, for a total of 2 to 4 times.

9. The method according to claim 7, characterized in that: When the angelica seedlings are soaked, the effective live bacteria concentration of the bacterial agent is 2×10 6 ~2×10 7 CFU / mL, the immersion time is 30 to 60 minutes.

10. The method according to claim 7, characterized in that When the foliage of the angelica seedlings in the cultivation period is sprayed, the spraying amount each time is 4×10 10 ~8×10 11 CFU / mu, spray once every 2 weeks, for a total of 2 to 4 times.

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