<i>Bacillus velezensis </i>strain for preventing and controlling early bolting of <i>Angelica sinensis</i>, microbial agent and use thereof

Abstract

A strain separation method for preventing and controlling early bolting of Angelica sinensis, and preparation of a microbial agent and use thereof, the strain is obtained by separation, purification and cultivation from rhizosphere soil of Angelica sinensis, and is identified as Bacillus spp. by Microbial 16S rDNA sequencing; the strain was deposited at China Center for Type Culture Collection on Jun. 24, 2021 under CCTCC NO: M 2021767; tThe Bacillus velezensis XG3 strain provided by the present invention can promote the seed germination of Angelica sinensis and delay the flowering of Arabidopsis thaliana for about 2 days; according to field test verification, the microbial agent can increase the root weight, root diameter and rootlet number of Angelica sinensis, can effectively prevent and control early bolting of Angelica sinensis.

Claims

1. A method for preventing and controlling early bolting of Angelica sinensis, comprising a step of administering Bacillus velezensis XG3 on the Angelica sinensis, wherein the Bacillus velezensis XG3 deposited at China Center for Type Culture Collection (CCTCC) under CCTCC NO: M 2021767.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a front view of a colony of Bacillus velezensis XG3 strain;

(2) FIG. 2 is a back view of a colony of Bacillus velezensis XG3 strain;

(3) FIG. 3 shows the effect of Bacillus velezensis XG3 on flowering of Arabidopsis thaliana;

(4) FIG. 4 shows an experiment for screening protectants prepared from a microbial agent of Bacillus velezensis XG3;

(5) FIG. 5 shows the experimental results of protectants of the microbial agent of Bacillus velezensis XG3 by central composite design;

(6) FIG. 6 shows a microbial agent of Bacillus velezensis XG3; and

(7) FIG. 7 shows the effect of the microbial agent of Bacillus velezensis XG3 on the growth of Angelica sinensis.

DETAILED DESCRIPTION

(8) The present invention can be better understood according to the following examples. However, it is easily understood by those skilled in the art that the specific fermentation culture process and functional evaluation described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

(9) The chemical reagents used in the following examples are all commercially available from conventional sources.

Example 1

(10) A new Bacillus velezensis XG3 strain was obtained by separation according to the following method: (1) oscillation: stripping soil around the root of Angelica sinensis, shaking off and collecting rhizosphere surface soil, placing 5 g of the soil in a triangular flask containing 100 mL of sterile water, oscillating the flask on a thermostatic shaker at 37 C. for 2 h at 140 r/min, standing for 30 min to obtain a soil stock solution (10.sup.1), pipetting 1 mL of a supernatant by using a pipettor, adding the supernatant into 9 mL of sterile water, fully shaking and uniformly mixing the mixture to obtain a soil diluent (10.sup.2), and repeating the steps to obtain soil diluents (10.sup.1-10.sup.6); (2) separation: pipetting 0.1 mL of the soil diluents (10.sup.4, 10.sup.5 and 10.sup.6), and inoculating the soil diluents into a prepared LB solid culture medium (composed of 10 g/L peptone, 5 g/L yeast extract, and 10 g/L NaCl), with 3 replicates per dilution; inverting the plate, culturing the plate at 30 C., observing growth conditions of colonies at any time, inoculating bacteria with different forms in an LB solid culture medium by using an inoculating loop, and repeatedly purifying until a single colony is obtained; and (3) cultivation and preservation: picking the bacteria purified on the plate into a triangular flask containing an LB liquid culture medium, oscillating the flask on a thermostatic shaker at 37 C. for 24 h at 140 r/min, mixing the bacteria with 50% glycerol at a ratio of 1:1, then placing the mixture into a sterilized cryopreservation tube, uniformly mixing the mixture, sealing the tube by a sealing film, and storing the tube in an ultra-low temperature refrigerator at 80 C.

Example 2

(11) The new Bacillus velezensis XG3 strain obtained by separation was subjected to molecular identification by the following method:

(12) The Bacillus velezensis XG3 strain was fermented and cultured according to the method of Example 1, and the bacterial solution was sent to Sangon Biotech (Shanghai) Co., Ltd. for 16r DNA sequencing. The upstream primer was 27F (5-AGTTTGATCMTGGCTCAG-3 (SEQ ID NO: 1)), the downstream primer was 1492R (5-GGTTACCTTGTTACGACTT-3 (SEQ ID NO: 2)), and the 16Sr DNA sequences were aligned based on ribosomal database and analyzed through Blast sequence alignment analysis. As shown in Table 1, the XG3 strain had 100.0% homology to Bacillus velezensis, which was identified as Bacillus, and presumed to be Bacillus velezensis.

(13) The sequence of the strain was as follows:

(14) TABLE-US-00001 (SEQIDNO:3) GGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGG ACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACA CGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCT AATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCT TCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAG GTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATC GGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAG TAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTG AGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAA GTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCA CGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTT GTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTG ATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAA CTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGC GTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTA ACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCC TGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCG CCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACG GTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGT GGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTT GACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAG TGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGT TAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGT TGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGAT GACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACA ATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACA AATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCT GGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCG GGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAA GTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATG ATTGGGGTGAAGTCGT.

(15) TABLE-US-00002 TABLE 1 XG3 strain sequence alignment results Max Total Query E Per. Description Score Score Cover value Ident Accession Bacillus velezensis strain KKLW 2745 24640 100% 0.0 100.00% CP054714.1 chromosome, complete genome Bacillus velezensis strain B268 2745 24557 100% 0.0 100.00% CP053764.1 chromosome, complete genome Bacillus velezensis strain S4 2745 24629 100% 0.0 100.00% CP050424.1 chromosome, complete genome Bacillus velezensis strain UB2017 2745 24651 100% 0.0 100.00% CP049741.1 chromosome, complete genome Bacillus velezensis strain 2745 24553 100% 0.0 100.00% CP028961.1 SRCM102752 chromosome, complete genome Bacillus amyloliquefaciens strain 2745 24646 100% 0.0 100.00% CP065539.1 DGL1 chromosome, complete genome Bacillus velezensis strain KMU01 2745 24590 100% 0.0 100.00% CP063768.1 chromosome, complete genome Bacillus velezensis strain BSC16a 2745 24618 100% 0.0 100.00% CP062074.1 chromosome, complete genome Bacillus amyloliquefaciens strain 2745 24607 100% 0.0 100.00% CP061853.1 MOH1-5b chromosome, complete genome Bacillus velezensis strain BIOMA 2745 24540 100% 0.0 100.00% CP059318.1 BV10 chromosome, complete genome

Example 3

(16) The effect of the Bacillus velezensis XG3 strain on seed germination of Angelica sinensis was tested by the following method:

(17) 600 Angelica sinensis seeds with full seeds and uniform quality were preferably selected, every 150 seeds were put into 1 culture flask, the flasks numbered 1-4 were separately sterilized with 75% ethanol for 1 min, sterilized with mercury dichloride for 9 min, and rinsed with sterile water for 5 times, and the sterile water was poured out. The Bacillus velezensis XG3 bacterial solution obtained according to the fermentation culture method of Example 2 was diluted and measured for OD600 values, and 50 mL of the diluted XG3 bacterial solutions (OD600 values were 0.05, 0.5 and 1, respectively) were poured into the No. 1-3 flasks, respectively, and 50 mL of an LB liquid culture medium was poured into the No. 4 flask and immersed for 24 h. Then, the seeds were transferred to culture dishes with 2 layers of sterile filter paper, 30 seeds were placed in order in each culture dish, and 5 replicates were set for each sample; about 5 mL of sterile water was poured to fully saturate the seeds and the filter paper, and the culture dish was sealed by a sealing film to prevent pollution. The culture dish and the seeds were weighed together and recorded. Then the culture dish was placed in an illumination incubator for incubation, the culture dish was weighed every 24 h, the weight of the culture dish was made up to the original weight, and the germination number of the seeds was recorded. The contaminated seeds were removed in time by using sterile tweezers. The results are shown in Table 2, and the Bacillus velezensis XG3 bacterial solutions obtained by screening can promote seed germination of Angelica sinensis.

(18) TABLE-US-00003 TABLE 2 Short-term germination trend of Angelica sinensis seeds treated with XG3 bacterial solutions with different concentrations Final germination 11 d 12 d 14 d 15 d 17 d 20 d rate Control 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 59.52% group XG3-0.05 0.00% 1.35% 4.05% 5.48% 10.00% 20.00% 70.00% XG3-0.5 0.00% 8.43% 16.87% 37.33% 59.09% 65.15% 83.33% XG3-1 0.91% 7.00% 9.00% 17.00% 29.29% 39.39% 71.72%

Example 4

(19) The effect of Bacillus velezensis XG3 on flowering of Arabidopsis thaliana was investigated by the following method:

(20) A proper amount of Col-0 Arabidopsis thaliana seeds were taken and sterilized with 75% ethanol (v/v) for 5 min, sterilized with 5% sodium hypochlorite for 5 min, and then repeatedly washed with sterile water for 4-6 times, 1 min each time; the sterilized seeds were then placed into a MS culture medium, with 15-20 seeds per flask, to ensure uniform distribution among seeds and proper density for later growth and transplantation. When the Arabidopsis thaliana seedlings grew to 4-6 leaves, about 2-3 weeks after formal culture, the Arabidopsis thaliana seedlings with relatively consistent growth states were slowly taken out from the culture medium by using tweezers, the culture medium on the roots was removed, and the seedlings were transplanted into prepared matrix soil. The matrix soil was prepared by uniformly mixing peat soil, vermiculite and perlite at a ratio of 1:1:1, and sterilized at 121 C. for 15 min. After the seedlings were transferred into the matrix soil for adaptive culture for 3 days, the XG3 liquid strain was inoculated by adopting a root irrigation method, the seedlings were irrigated and inoculated once a week until they fully bloomed, and the growth state, the flowering time and the number of flowering rosette leaves of Arabidopsis thaliana were observed and recorded. The results are shown in FIG. 3: blank water was given to the control group, and there was a remarkable late flowering phenomenon when the Arabidopsis thaliana was treated with XG3, indicating that the XG3 strain has the potential to be developed as a microbial agent for preventing and controlling early bolting of Angelica sinensis.

Example 5: Screening Experiment for Composite Protectant

(21) The optimal composite protectant of the XG3 microbial agent was optimized through a single-factor test and a central composite design test method. (1) In the single-factor test, trehalose (mus, 0.5 mg/mL, 1 mg/mL and 2 mg/mL), sucrose (suc, 2.5 mg/mL, 5 mg/mL and 10 mg/mL), sodium glutamate (msg, 0.5 mg/mL, 1 mg/mL and 2 mg/mL) and glucose (glu, 5 mg/mL, 10 mg/mL and 15 mg/mL) were selected as protectants, three concentrations were separately set, sterile water was taken as a control, the survival rate of the lyophilized Bacillus velezensis was taken as an index, and the concentrations of the protectants with a good protective effect were selected. According to the screening results of the single-factor test, the proportion of trehalose, sucrose, sodium glutamate and glucose was optimized by adopting the response surface central composite design, and the optimal formula was determined by taking the survival rate of the lyophilized Bacillus velezensis as an index. (2) In order to increase the number of viable bacteria in the lyophilized powder of the microbial agent, various protectants were usually added before the bacteria were frozen to maintain the activity and stability of the strain. As could be seen from FIG. 4, the survival rate of the lyophilized bacteria was 20.12% when the bacteria were lyophilized with sterile water without the addition of any protectant, and the survival rate of the bacteria increased to various degrees with addition of different protectants. According to the results of the single-factor test, secondary regression fitting was carried out by using Design-Expert 11.0 software to obtain a fitting equation of the survival rate Y=88.74+5.10A3.82B+0.2176C+3.22D2.48A.sup.28.17B.sup.27.57C.sup.21.94D.sup.2. Analysis of variance (Table 3) showed that the fitted regression equation accorded with the above test principle, the adaptability was good, the optimal formula (FIG. 5) of the XG3 lyophilized protectant obtained by prediction analysis of a regression model was 15 mg/mL glucose, 5.37 mg/mL sucrose, 1.26 mg/mL trehalose and 1.87 mg/mL sodium glutamate, and the predicted survival rate was 87.96%. A verification test was carried out under the formula conditions, the survival rate of XG3 was 82.45%, and the difference between the measured value and the predicted value was small, indicating that the formula was feasible. As could be seen from FIG. 6, the color of the microbial agent powder prepared by lyophilizing the blank water was yellowish, and the color of the microbial agent powder prepared by lyophilizing the protectant was off-white; the volume of the lyophilized protectant was slightly different from that of the protectant before lyophilization, which slightly decreased, and the volume of the lyophilized blank water significantly decreased.

(22) TABLE-US-00004 TABLE 3 Analysis of variance of response surface model sum of mean F- P- source squares df square value value Model 526.79 8 65.85 7.86 0.03 significant A-glu 57.76 1 57.76 6.89 0.06 B-suc 37.75 1 37.75 4.51 0.10 C-mus 0.12 1 0.12 0.01 0.91 D-msg 26.76 1 26.76 3.19 0.15 AB 0 0 AC 0 0 AD 0 0 BC 0 0 BD 0 0 CD 0 0 A.sup.2 25.90 1 25.90 3.09 0.15 B.sup.2 176.34 1 176.34 21.04 0.01 C.sup.2 151.33 1 151.33 18.06 0.01 D.sup.2 9.96 1 9.96 1.19 0.34 Residual 33.52 4 8.38 Cor Total 560.31 12 R.sup.2 0.9402 CV % 8.83 Adeq 9.4299 Precision

Example 6

(23) This example relates to a preparation method for a microbial agent of a Bacillus velezensis XG3 strain comprising the following steps: (1) The Bacillus velezensis XG3 strain was inoculated in an LB liquid culture medium and cultured for activation on a thermostatic shaker at 37 C. for 24 h at 140 r/min. The strain activated overnight was transferred at a ratio of 1% (v/v) to a new LB liquid culture medium and cultured at 140 r/min for 24 h. The OD value was measured. The fermentation liquor was centrifuged at 4000 rpm for 5 min, then the supernatant was discarded. The bacterial precipitate was collected. (2) 1 mL of a composite protectant (15 mg/mL glucose, 5.37 mg/mL sucrose, 1.26 mg/mL trehalose, and 1.87 mg/mL sodium glutamate) solution was added into the bacterial precipitate obtained by centrifugation per 100 mL of the bacterial solution. The sample was pre-frozen in an ultra-low temperature refrigerator at 80 C. After the pre-freezing was completed, the sample was quickly transferred into a vacuum lyophilizer and lyophilized at 40 C. and 10 Pa. Finally, the lyophilized substance was ground, namely lyophilized in vacuum, to obtain a lyophilized XG3 powder microbial agent.

Example 7. The Effects of the Microbial Agent of Bacillus velezensis XG3 on the Growth of Angelica sinensis and Early Bolting Prevention and Control were Verified by an Experiment on Test Field Samples

(24) The Angelica sinensis seedlings not treated by root irrigation were preferred. Blank water was given to the control group at 500 mL per ridge, while the treatment groups received a low-dose at 0.8 g per ridge (XG3L), a medium-dose at 2.4 g per ridge (XG3M), or a high-dose at 7.2 g per ridge (XG3H) of the XG3 microbial agent prepared by the method of Example 6. A total of three interventions were given once every two weeks since one week after the Angelica sinensis seedlings turned green. The first intervention was given by root irrigation at 5 mL per plant, and the other two were given by spraying. The effect of the XG3 microbial agent on bolting and flowering and quality formation of Angelica sinensis was monitored. The early bolting rate of Angelica sinensis in the blank control group was 32.03%, and the early bolting rates of XG3 were 18.75%, 22.27% and 11.72% in the low-dose group, the medium-dose group and the high-dose group, respectively, indicating that the XG3 microbial agent prepared by the present invention could effectively prevent and control the early bolting of Angelica sinensis. In addition, as shown in FIG. 7, the microbial agent prepared by the present invention could also significantly increase the yield of Angelica sinensis, the diameter of a main root and the number of rootlets.

(25) The above results show that Bacillus velezensis XG3, a new angelica rhizosphere bacterium, is obtained by screening in the present invention, and the separation, culture, storage and growth promotion function analysis thereof and the preparation method for a microbial agent thereof are established, the Bacillus velezensis XG3 has the good functions of promoting seed germination and plant growth of Angelica sinensis and delaying the flowering of Arabidopsis thaliana, and the microbial agent developed and prepared therefrom can effectively prevent and control early bolting of Angelica sinensis and increase the yield of Angelica sinensis, and has important practical application values.

(26) The above descriptions are only preferred embodiments of the present invention. It should be noted that those of ordinary skill in the art can also make several improvements and modifications without departing from the principle of the present invention, and such improvements and modifications shall fall within the protection scope of the present invention.