CN114045242A - Bacillus belgii XG2 strain for producing phthalides and application thereof - Google Patents
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Abstract
The invention relates to a Bacillus beiLeisi XG2 strain for producing phthalide components and application thereof, the strain is obtained by separating, purifying and culturing angelica rhizosphere soil, and is identified as bacillus by microorganism 16S rDNA sequencing. The strain is preserved in China center for type culture Collection with the preservation number: CCTCC NO: M2021766. The invention provides a Bacillus beiLeisi strain with stable character, simple culture medium and good growth for preparing phthalide components by large-scale fermentation, and establishes a practical and simple fermentation culture method and a separation and extraction process thereof, and the components of the fermentation liquor after extraction and concentration can be used as a new source of the active components of angelica. The Bacillus belgii XG2 has effects of promoting germination of radix Angelicae sinensis seed, and increasing plant height and fresh weight of radix Angelicae sinensis seedling.
Description
Technical Field
The invention relates to a Bacillus beleisi strain, which can produce phthalide components and promote germination of angelica sinensis seeds and plant growth, and belongs to the technical field of microorganisms.
Background
The structure and composition of the bacterial community in the soil can effectively reflect the health condition of a soil ecosystem, the productivity of the soil and the degree of environmental interference, and the change of the population quantity and the structure of rhizosphere microorganisms can cause the change of the function of the microbial community. Plant growth-promoting bacteria (PGPRs) are a group of self-growing bacteria distributed on the surface of a Plant root system in soil around the root system, and affect the structure of the Plant root system by secreting relevant Plant hormones or hormone precursor substances. PGPR promotes the secretion of plant hormones such as indoleacetic acid, abscisic acid, cytokinin, gibberellin, ethylene and the like through the functions of nitrogen fixation, phosphorus dissolution, potassium dissolution, iron dissolution and the like, and has the functions of promoting the growth of plants and improving the stress resistance of the plants. When the exogenous microbial agent is added, the ecological level of soil microbes can be changed, so that the content of rhizosphere soil nutrients and the nutrient absorption and utilization capacity of plants are improved, and the quality and the yield of the angelica are improved.
Meanwhile, rhizobacteria have a strong metabolic function, and can produce active ingredients the same as or similar to those of host plants in addition to nutrients, phytohormones and the like. The variety of the species of rhizosphere bacteria endows the metabolites with the variety, the rhizosphere bacteria fermentation method has great potential for mining new resources, and the rhizosphere bacteria capable of metabolizing to produce corresponding active ingredients are utilized to produce novel, efficient and low-cost active drugs, so that the current situation of shortage of rare traditional Chinese medicine plant resources can be effectively relieved.
Angelica sinensis (Oliv.) Diels, radix Angelicae sinensis, is the dried root of Angelica sinensis (Oliv.) Diels of Umbelliferae, and is distributed in Gansu, Sichuan and Yunnan provinces, and has effects of replenishing blood, promoting blood circulation, regulating menstruation, relieving pain, and loosening bowel to relieve constipation. The main active substances of the angelica comprise volatile oil, organic acid, polysaccharide, flavonoid, amino acid, trace elements and the like. Wherein the volatile oil mainly contains ligustilide, n-butylphthalide, butenyl phthalide, senkyunolide, etc. Clinical studies show that the phthalides have good pharmacological activity in the aspects of smooth muscle system, cardiovascular system, immune system, nervous system and the like. However, the angelica has long growth period, slow growth speed, strong dependence on producing areas, serious damage to wild resources in the land, easy occurrence of early bolting, plant diseases and insect pests in the cultivation process and the like, and seriously restricts the quick and benign development of the angelica industry. At present, some scholars research the microbial resources of angelica sinensis, but the number and the types of strains are limited, the function screening research is weak, new strains for producing the active ingredients of angelica sinensis and promoting the high-quality growth of angelica sinensis are further screened, and the method has important significance for deeply developing the microbial fermentation for producing phthalide ingredients, searching for novel phthalide ingredient sources, relieving the current situation of shortage of angelica sinensis resources, developing biological bacterial fertilizers, developing green and healthy planting and the like.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a Bacillus belgii XG2 strain for producing phthalide components, and a separation method and a fermentation culture method of the strain. Another purpose of the invention is to provide the analysis of the fermentation product of the strain and the application of the fermentation product in the angelica sinensis seed germination and plant growth processes.
The technical scheme is as follows: in order to realize the purpose, the invention adopts the technical scheme that:
the Bacillus beiLeisi XG2 for producing phthalide components provided by the invention is obtained by separating, purifying and culturing the strain from angelica rhizosphere soil, and is identified as Bacillus (Bacillus spp.) by microorganism 16S rDNA sequencing. The strain is preserved in China center for type culture Collection with the preservation number: CCTCC NO: M2021766; the 16S rDNA Sequence of this strain is shown in Sequence listing.
The invention provides a method for separating a Bacillus belgii XG2 strain, which comprises the steps of oscillating, inoculating, separating, culturing and storing, and comprises the following specific steps:
a) oscillating: stripping soil around radix Angelicae sinensis root, shaking off to collect rhizosphere surface soil, placing 5g of soil in 100mL triangular flask in sterile water, shaking at 30 deg.C with constant temperature shaking table 160r/min for 2 hr, standing for 30min to obtain soil stock solution (10)-1)，Then using a pipette to suck 1mL of supernatant fluid, adding the supernatant fluid into 9mL of sterile water, and fully shaking up to obtain the (10)-2) By analogy, 10 is obtained-1-10-6And (4) diluting the solution.
b) Separation: 0.1mL of 10 was aspirated-4、10-5、10-6The diluted soil solution is inoculated into the prepared LB solid culture medium, and each dilution is set to be 3 times. Inverting the plate, culturing at 30 deg.c and observing colony growth, inoculating bacteria of different forms to LB solid culture medium with inoculating loop, and repeated purifying until obtaining single colony.
c) Culturing and preserving: picking the bacteria purified on the plate into a triangular flask filled with LB liquid culture medium, shaking for 24h at 30 ℃ with a constant temperature shaking table at 160r/min, mixing with 50% glycerol 1: 1, placing the mixture into a sterilized cryopreservation tube, mixing the mixture evenly, sealing the tube with a sealing film, and storing the tube in an ultra-low temperature refrigerator at minus 80 ℃.
The fermentation culture method of the Bacillus belgii XG2 strain provided by the invention comprises the following steps: inoculating Bacillus velezensis XG2 strain in 50mLLB liquid culture medium, performing activation culture at 30 ℃ at constant temperature by 160r/min for 24h, taking 1mL of activated strain to a triangular flask filled with 500mL of LB liquid culture medium for fermentation, and performing shake culture at 30 ℃ at 160r/min for 24 h; after the fermentation culture, collecting fermentation liquor.
Preferably, in the fermentation method of Bacillus belgii, the LB medium consists of 10g/L peptone, 5g/L yeast extract and 10g/L NaCl.
Analysis of fermentation products of the bacillus beleisi XG2 strain, taking fermentation liquor obtained by the fermentation method as a raw material, extracting with ethyl acetate for three times, volatilizing the ethyl acetate, redissolving with methanol, detecting the fermentation products of the solution, and detecting to find that the solution contains three phthalide components: ligustilide, n-butylphthalide, and butenyl phthalide.
The application of the Bacillus beilesiensis XG2 strain in the germination and plant growth process of angelica sinensis seeds is characterized in that fermentation liquor is obtained by the fermentation method, diluted into bacterial solutions with different OD concentrations and co-cultured with the angelica sinensis seeds and the angelica sinensis aseptic seedlings.
Has the advantages that: compared with the existing research, the invention has the following advantages:
(1) the Bacillus beleisi XG2 strain provided by the invention can be directly metabolized to generate phthalide components such as ligustilide, n-butylphthalide, butenyl phthalide and the like, has important significance for deeply developing the research of producing the phthalide components by microbial fermentation, and provides a new source for the active components of angelica.
(2) The Bacillus beilesiensis XG2 strain provided by the invention can promote germination of angelica sinensis seeds, increase plant height and fresh weight of angelica sinensis seedlings, can be used for developing biological bacterial fertilizers which are low in cost, pollution-free, stable in yield increase and capable of effectively improving soil, and further can be applied to the green and healthy planting industry of angelica sinensis.
Drawings
FIG. 1 front side of a colony of Bacillus belgii XG2 strain;
FIG. 2 the back of the colony of Bacillus belgii XG2 strain;
FIG. 3 is a total ion diagram of six phthalide component standards; the retention time of the components in the figure is ligustilide of 12.67 min; butenyl phthalide 12.81 min; 11.55min for n-butylphthalide; senkyunolide A, 11.15 min; senkyunolide H for 4.99 min; senkyunolide I, 4.76 min);
FIG. 4 is a total ion diagram of phthalide components of a sample of Bacillus belgii XG2 strain;
FIG. 5 chromatogram of ligustilide standard;
FIG. 6 depicts total ligustilide ion graphs (TIC) and extracted ion graphs (EIC) of a sample of Bacillus beilesiensis XG2 strain;
FIG. 7 Total ion diagram (TIC) and extracted ion diagram (EIC) of n-butylphthalide standard;
FIG. 8 is a sample of Bacillus belgii XG2 strain n-butylphthalide total ion map (TIC) and extracted ion map (EIC);
FIG. 9 is a schematic diagram of a total ion diagram (TIC) and an extracted ion diagram (EIC) of a butenyl phthalide standard;
FIG. 10 sample butenyl phthalide total ion map (TIC) and extracted ion map (EIC) of Bacillus belgii XG2 strain;
FIG. 11 the growth effect of B.beilesiensis XG2 strain on Angelica sinensis.
Detailed description of the preferred embodiments
The invention will be better understood from the following examples. However, it is readily understood by those skilled in the art that the specific fermentation culture processes and functional evaluations described in the examples are only illustrative of the present invention and should not, nor should they be taken as limiting the invention as detailed in the claims.
Example 1
A new Bacillus belgii XG2 strain is separated by the following method:
(1) oscillating: stripping soil around radix Angelicae sinensis root, shaking off to collect rhizosphere surface soil, placing 5g of soil in 100mL triangular flask in sterile water, shaking at 30 deg.C with constant temperature shaking table 160r/min for 2 hr, standing for 30min to obtain soil stock solution (10)-1) Then sucking 1mL of supernatant with a pipette gun, adding into 9mL of sterile water, and shaking thoroughly to obtain (10)-2) By analogy, 10 is obtained-1-10-6And (4) diluting the solution.
(2) Separation: 0.1mL of 10 was aspirated-4、10-5、10-6The diluted solution was inoculated into a prepared LB solid medium (10g/L peptone, 5g/L yeast extract, 10g/L NaCl) in 3 replicates per dilution. Inverting the plate, culturing at 30 deg.c and observing colony growth, inoculating bacteria of different forms to LB solid culture medium with inoculating loop, and repeated purifying until obtaining single colony.
(3) Culturing and preserving: picking the bacteria purified on the plate into a triangular flask filled with LB liquid culture medium, shaking for 24h at 30 ℃ with a constant temperature shaking table at 160r/min, mixing with 50% glycerol 1: 1, placing the mixture into a sterilized cryopreservation tube, mixing the mixture evenly, sealing the tube with a sealing film, and storing the tube in an ultra-low temperature refrigerator at minus 80 ℃.
Example 2
A new Bacillus belgii XG2 strain is cultured by the following fermentation method:
(1) inoculating a single colony obtained by repeated purification in the step (2) in the embodiment 1 into a 50mLLB liquid culture medium, performing activation culture at 30 ℃ for 160r/min by using a constant temperature shaking table, after 24 hours, taking 1mL of activated strain to a triangular flask filled with 500mL of LB liquid culture medium for fermentation, and performing shaking culture at 30 ℃ for 160r/min for 24 hours; after the fermentation culture, collecting fermentation liquor.
(2) LB medium involved in the isolation and fermentation of the strains consisted of 10g/L peptone, 5g/L yeast extract, 10g/L NaCl.
Example 3
A new Bacillus belgii XG2 strain is obtained by analyzing and screening the following metabolites:
(1) extracting the fermentation liquor sample obtained in the example 2 with equal amount of ethyl acetate for three times, volatilizing the ethyl acetate, redissolving the methanol, detecting by using an ultra-high performance liquid chromatography-triple quadrupole composite linear ion TRAP mass spectrometry (UPLC-Q-TRAP/MS) combined method, and comparing the strain fermentation ion graph with a standard substance to determine whether the fermentation liquor contains phthalide components.
(2) Mixing six kinds of phthalide components ligustilide, n-butylphthalide, butenyl phthalide and senkyunolide A, H, I with abundant content in radix Angelicae sinensis, and performing LC-MS analysis, wherein the six kinds of standard Total ion graphs (Total ion chromatography), ligustilide, n-butylphthalide and butenyl phthalide standard extracted ion graphs (Extract ion chromatography) are shown in FIGS. 3, 5, 7 and 9.
(3) The metabolites obtained in the step (1) of example 3 were subjected to LC-MS (liquid chromatography-Mass Spectrometry) analysis, and FIG. 4, FIG. 6, FIG. 8 and FIG. 10 show Total ion maps (Total ion chromatography) of the metabolites of Bacillus beijerinckii XG2 strain, ligustilide, n-butylphthalide and butenyl phthalide extracted ion maps (Extract ion chromatography) in the strain, respectively.
(4) From fig. 3 to fig. 10, the metabolite of the bacillus belgii XG2 strain contains ligustilide, n-butylphthalide, and butenyl phthalide. The stored bacillus belgii XG2 strain was subjected to fermentation culture and fermentation broth treatment according to the methods of examples 2 and 3 to obtain another 6 samples of metabolites of the bacillus belgii XG2 strain, and the results of the chromatography analysis by mass spectrometry showed that the samples all contained ligustilide, n-butylphthalide and butenyl phthalide.
Example 4
A new Bacillus belgii XG2 strain is molecularly identified by the method:
bacillus belgii XG2 was cultured by fermentation according to the method of example 2, and the resulting bacterial liquid was sent to Biotechnology (Shanghai) Ltd for 16rDNA sequencing. The upstream primer 27F (5 '-AGTTTGATCMTGGCTCAG-3'), the downstream primer 1492R (5'-GGTTACCTTGTTACGACTT-3'), and the 16SrDNA sequence in ribosome database: (http:// rdp.cme.msu.edu/index.jsp) In the comparison, through Blast sequence comparison analysis, the homology of the XG2 strain and the Bacillus belgii is 100.0%, and the strain is identified as Bacillus (Bacillus), and is presumed to be Bacillus velezensis (Bacillus belgii).
Sequence listing
GGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGT
TABLE 1 XG2 Strain sequence alignment results Table
Example 5 Bacillus belgii XG2 Strain Germination assay on Angelica sinensis seeds
Preferably 600 full and uniform-quality angelica seeds are selected, every 150 angelica seeds are put into 1 culture bottle, the label 1-4 is sterilized by 75 percent ethanol for 1min, mercuric chloride is sterilized for 9min, sterile water is rinsed for 5 times, and the sterile water is poured out. Bacillus belgii XG2 was obtained according to the fermentation culture method of example 2, diluted and measured for OD600, 50mL of XG-2 diluted solutions with OD600 of 0.05, 0.5, and 1 were poured into No. 1-3 flasks, 50mL of liquid medium was poured into No. 4 flasks, and the flasks were soaked for 24 hours. Then, the seeds are transferred to culture dishes filled with 2 layers of sterile filter paper, 30 seeds are placed in order in each culture dish, 5 treatments are repeated on each sample, about 5mL of sterile water is poured to fully saturate the seeds and the filter paper, and the culture dishes are sealed by a sealing film to prevent pollution. The petri dish and seed were weighed together and recorded. The above dishes were then placed in a light incubator for cultivation, the dishes were weighed every 24h and made up to the original weight and the number of seeds germinated was recorded. The contaminated seeds are removed in time by using a pair of disinfecting tweezers. The results are shown in table 2, and the bacillus beilesensis XG2 bacterial liquid obtained by screening can promote germination of angelica sinensis seeds.
Table 2 short-term germination vigor of angelica sinensis seeds treated by XG2 bacteria solutions with different concentrations
| 10d | 11d | 13d | 14d | 16d | 19d | |
| control | 0.00％ | 0.00％ | 0.00％ | 0.00％ | 0.00％ | 0.00％ |
| XG-2-0.05 | 0.00％ | 0.00％ | 0.00％ | 1.33％ | 1.48％ | 2.00％ |
| XG-2-0.5 | 0.00％ | 1.50％ | 3.33％ | 4.67％ | 8.00％ | 10.67％ |
| XG-2-1 | 0.67％ | 0.72％ | 1.33％ | 4.67％ | 8.57％ | 10.67％ |
。
Example 6 test of the Effect of Bacillus beilesiensis XG2 Strain on Angelica sinensis growth
Selecting full and uniform-quality angelica seeds, sequentially sterilizing the angelica seeds with 75% ethanol for 1min, sterilizing with mercuric chloride for 9min, rinsing with sterile water for 5 times, pouring out the sterile water, uniformly placing the sterilized angelica seeds in an MS culture medium, placing 5 seeds in each bottle, and placing the inoculated culture bottles into an artificial climate box for culture at the culture temperature of 23 ℃, the humidity of 70%, the illumination time of 14h/d and the illumination intensity of 2000 lx. And (3) taking the angelica sinensis seedlings which grow true leaves and are relatively uniform in size after being cultured for about 30 days, randomly grouping the angelica sinensis seedlings into a blank group and an XG-2 group, wherein each group comprises three bottles, six angelica sinensis seedlings are placed in each bottle, and 20 mu L of blank LB culture medium or XG-2 bacterial liquid with OD600 of 0.5 is added to the root of each seedling. Co-culturing the bacteria solution and the sterile seedlings for 7 days, and taking the angelica sterile seedlings to measure fresh weight and plant height. As shown in FIG. 11, the bacterial liquid of Bacillus beilesiensis XG2 obtained by screening in the invention can promote the growth of Angelica sinensis and increase its fresh weight.
The results show that the new angelica rhizosphere bacterium, namely the Bacillus belgii XG2, is obtained by screening, and a method for separating, fermenting, culturing, storing, identifying metabolites and analyzing growth promoting functions of the new angelica rhizosphere bacterium is established, so that the Bacillus belgii XG2 strain can be directly metabolized to generate angelica pharmacodynamic active ingredients, namely ligustilide, n-butylphthalide and butenyl phthalide, and a new ingredient source is provided for the preparation of medical raw materials and functional products. Meanwhile, the Bacillus beilesiensis XG2 has the functions of promoting angelica seed germination and angelica plant growth, can be used for developing related products such as biological bacterial manure, and has important practical application value.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (7)
1. A Bacillus belgii for producing phthalides is preserved in China Center for Type Culture Collection (CCTCC), the preservation number is M2021766, and the Bacillus velezensis XG2 is named in classification.
2. A method for fermentation by using Bacillus velezensis XG2 is characterized in that the strain of Bacillus velezensis XG2 as defined in claim 1 is inoculated in LB liquid culture medium, and is subjected to constant temperature shaking table activation culture, and then the activated strain is taken to be put into a triangular flask filled with the LB liquid culture medium for fermentation and shaking table culture; collecting the fermentation liquor.
3. The method for fermentation by Bacillus velezensis XG2 according to claim 2, wherein the Bacillus velezensis XG2 strain of claim 1 is inoculated into LB liquid medium, activated and cultured at 30 ℃ for 160r/min, 1mL of activated strain is taken out to be put into a triangular flask containing 500mL of LB liquid medium for fermentation after 24h, and cultured at 30 ℃ for 160r/min for 24 h; after the fermentation culture, collecting fermentation liquor.
4. The method for fermentation using Bacillus belgii as claimed in claim 2 or 3, wherein the LB medium is composed of 10g/L peptone, 5g/L yeast extract, 10g/L NaCl.
5. The method according to claim 2 or 3, wherein the fermentation broth comprises three phthalide components, i.e., ligustilide, n-butylphthalide and butenyl phthalide.
6. The application of the Bacillus belgii of claim 1 in preparing a culture agent for promoting germination of angelica sinensis seeds and improving plant height and fresh weight of angelica sinensis seeds.
7. The use of the fermentation broth of bacillus belgii of claim 1 or 2 in the preparation of a culture solution for promoting germination of angelica sinensis seeds and increasing plant height and fresh weight of angelica sinensis seeds.
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| CN110982725A (en) * | 2018-09-29 | 2020-04-10 | 福建省农业科学院农业生物资源研究所 | Bacillus for antagonizing fusarium wilt and promoting growth and application thereof |
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| CN111254086A (en) * | 2019-12-27 | 2020-06-09 | 华中农业大学 | Bacillus belgii and application thereof in biocontrol |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110982725A (en) * | 2018-09-29 | 2020-04-10 | 福建省农业科学院农业生物资源研究所 | Bacillus for antagonizing fusarium wilt and promoting growth and application thereof |
| CN110982724A (en) * | 2018-09-29 | 2020-04-10 | 福建省农业科学院农业生物资源研究所 | Bacillus for antagonizing phytopathogen and promoting rooting and application thereof |
| CN111254086A (en) * | 2019-12-27 | 2020-06-09 | 华中农业大学 | Bacillus belgii and application thereof in biocontrol |
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| Title |
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| WEI-MENG FENG ET AL: "Impact of Bacillus on Phthalides Accumulation in Angelica sinensis (Oliv.) by Stoichiometry and Microbial Diversity Analysis", FRONTIERS IN MICROBIOLOGY, vol. 11, pages 1 - 10 * |
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