CN116121144B - Atractylodes macrocephala growth promoting bacterium BZ-29 and application thereof - Google Patents
Atractylodes macrocephala growth promoting bacterium BZ-29 and application thereof Download PDFInfo
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Abstract
The invention provides a bighead atractylodes rhizome growth-promoting bacterium BZ-29 and application thereof, and belongs to the technical field of microorganisms. The invention provides bacillus amyloliquefaciens BZ-29, and the preservation number of the bacillus amyloliquefaciens BZ-29 is CCTCCM20221991. The bacillus amyloliquefaciens BZ-29 provided by the invention can promote the increase of the stem length of bighead atractylodes rhizome by 91.67%, the increase of root length by 88.24%, the increase of fresh weight by 175.99% and the increase of dry weight by 132.50%; meanwhile, the bacillus amyloliquefaciens BZ-29 has the effect of promoting the bighead atractylodes rhizome to synthesize sesquiterpene lactone, and has remarkable promoting effect on the expression of the bighead atractylodes rhizome to synthesize sesquiterpene lactone key gene DXR, FPPS, HMGR. The discovery of the bighead atractylodes rhizome growth-promoting bacterium BZ-29 lays a good foundation for developing and utilizing bighead atractylodes rhizome growth-promoting bacterial manure.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a bighead atractylodes rhizome growth-promoting bacterium BZ-29 and application thereof.
Background
Atractylodis rhizoma, one of the common bulk medicinal materials, belonging to the genus Atractylodes of Compositae, is used as a root and stem medicine, has the effects of invigorating spleen, replenishing qi, eliminating dampness and promoting diuresis, is one of the eight ingredients of Zhejiang, has the theory of 'ten-medicine nine-operation', and is mainly planted in Zhejiang in the areas of Zhi, xinchang, hebei Anguo, anhui, hubei and the like.
The bighead atractylodes rhizome grows slowly under natural conditions. In order to pursue the yield of the bighead atractylodes rhizome and improve the disease resistance of the bighead atractylodes rhizome, a large amount of fertilizers, pesticides and the like are often applied, and the problems of soil acidification, water pollution, unbalanced soil micro-ecological structure and the like caused by abuse of the fertilizers and the pesticides are not beneficial to sustainable development of the bighead atractylodes rhizome industry. In order to improve the yield of the bighead atractylodes rhizome, the biological bacterial agent for promoting the growth of the bighead atractylodes rhizome is obtained through separation and screening, and is developed into biological bacterial fertilizer to be applied, so that the biological bacterial fertilizer has important significance for the development of the bighead atractylodes rhizome industry.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a biological microbial agent capable of promoting the growth of bighead atractylodes rhizome.
The invention aims at realizing the following technical scheme:
the invention provides bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BZ-29, and the preservation number of the bacillus amyloliquefaciens BZ-29 is CCTCC M20221991.
Preferably, the 16S rDNA sequence of the bacillus amyloliquefaciens BZ-29 is shown in SEQ ID NO. 1.
The invention provides the fermentation broth of the bacillus amyloliquefaciens BZ-29.
The invention provides a preparation method of the fermentation broth, which comprises the following steps:
culturing the bacillus amyloliquefaciens BZ-29 in a culture medium to obtain fermentation broth.
The invention provides application of the bacillus amyloliquefaciens BZ-29 or the zymophyte liquid in promoting plant growth and/or preventing and controlling diseases.
Preferably, the plant comprises any one or more of bighead atractylodes rhizome, corn and tobacco.
Preferably, the promotion of plant growth includes any one or two or more of the following (1) to (4):
(1) Promoting the absorption of nutrient elements by plants;
(2) Promoting stem growth;
(3) Promote root elongation;
(4) The dry weight and fresh weight of the plants are improved.
Preferably, the disease control comprises the control of peanut southern blight germ, rice rhizoctonia solani, wheat take-all germ and rice sheath blight germ 、 Caused by one or more fungi of wheat stem rot germ and sclerotinia rot germPlant diseases.
The invention provides application of bacillus amyloliquefaciens BZ-29 or fermentation broth in promoting synthesis of sesquiterpene lactone from bighead atractylodes rhizome.
The invention also provides a using method of the bacillus amyloliquefaciens BZ-29 or the fermentation broth in the technical scheme, and the application is carried out in the plant seed white-out period and/or when plants grow to 5-7 leaves; the application amount is 1-5 mL/plant.
The invention has the beneficial effects that:
the invention provides bacillus amyloliquefaciens BZ-29, and the preservation number of the bacillus amyloliquefaciens BZ-29 is CCTCC M20221991. The invention screens a bacterial strain BZ-29 which has the characteristics of auxin production, phosphate dissolution, cellulase production, siderophore production and antagonism of various pathogenic fungi from a large number of bighead atractylodes rhizome endophytic bacteria and has the growth promoting function on host bighead atractylodes rhizome, and is identified as bacillus amyloliquefaciens (Bacillus amyloliquefaciens). The IAA production capacity can reach 8.48 mug/mL, the phosphate dissolving content can reach 3.47mol/L by quantitative determination, the capability of producing siderophores is achieved, and the activity is more than 90%. The bacillus amyloliquefaciens (namely bighead atractylodes rhizome growth promoting bacteria) BZ-29 provided by the invention can promote the growth of bighead atractylodes rhizome in the seedling stage, and has the effect of promoting the bighead atractylodes rhizome to synthesize sesquiterpene lactone. The results of the examples show that: the bacillus amyloliquefaciens BZ-29 provided by the invention can promote the increase of the stem length of bighead atractylodes rhizome by 91.67%, the increase of root length by 88.24%, the increase of fresh weight by 175.99% and the increase of dry weight by 132.50%; the bacillus amyloliquefaciens BZ-29 has a remarkable promoting effect on the expression of the synthetic sesquiterpene lactone key gene DXR, FPPS, HMGR. The discovery of the bighead atractylodes rhizome growth-promoting bacterium BZ-29 lays a good foundation for developing and utilizing bighead atractylodes rhizome growth-promoting bacterial manure.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a colony morphology diagram of BZ-29 strain on LB, GS, PDA, V, OA solid medium;
FIG. 2 is a diagram showing the bacterial morphology of BZ-29 strain observed under a scanning electron microscope;
FIG. 3 is a molecular identification cluster map spectrum of BZ-29 strain;
FIG. 4 is a diagram of IAA production by BZ-29 strain;
FIG. 5 is an IAA standard graph;
FIG. 6 is a standard graph of phosphorus content;
FIG. 7 is a map of phosphate solubilizing of BZ-29 strain;
FIG. 8 is a diagram showing cellulase production by BZ-29 strain;
FIG. 9 is a chart of siderophore production capacity of BZ-29 strain;
FIG. 10 is a chart of BZ-29 strain antagonistic pathogenic fungi;
FIG. 11 is a graph showing the results of physiological and biochemical experiments of BZ-29 strain;
FIG. 12 is a graph showing the growth promotion effect of BZ-29 strain on the growth of Atractylodis rhizoma;
FIG. 13 is a graph showing the growth promotion effect of BZ-29 strain on the growth of Atractylodis rhizoma;
FIG. 14 is a graph showing the growth promotion of BZ-29 strain on tobacco growth;
FIG. 15 is a graph showing the result of the growth promotion of the BZ-29 strain on tobacco growth;
FIG. 16 is a graph showing the growth promotion of BZ-29 strain on maize growth;
FIG. 17 is a graph showing the results of the growth promotion of BZ-29 strain on maize growth;
FIG. 18 is a graph showing the effect of BZ-29 strain on the key gene of sesquiterpene synthesis from Atractylodis rhizoma.
Detailed Description
The invention provides bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BZ-29, and the preservation number of the bacillus amyloliquefaciens BZ-29 is CCTCC M20221991.
The bacillus amyloliquefaciens BZ-29 is separated from the white atractylodes rhizome and is endophyte of bighead atractylodes rhizome. The bacillus amyloliquefaciens BZ-29 of the invention is preserved in China center for type culture collection (China center for type culture collection) at 12 and 19 of 2022.
In the present invention, the culture medium of Bacillus amyloliquefaciens BZ-29 may be selected from LB medium, GS medium, PDA medium or V8 medium, but not OA medium. The morphological characteristics of the bacillus amyloliquefaciens BZ-29 are shown in figure 1, and specifically comprise the following steps: the bacillus amyloliquefaciens BZ-29 is light gray, has sticky surface and is smooth or wrinkled and has bulges on an LB solid culture medium; the bacillus amyloliquefaciens BZ-29 is bright white on a GS solid culture medium, has a thick and smooth surface and is provided with bulges; the bacillus amyloliquefaciens BZ-29 is gray white, has a sticky and smooth surface and is provided with obvious bulges on a PDA solid culture medium; the bacillus amyloliquefaciens BZ-29 is light off-white, has a sticky surface and is smooth or wrinkled and has obvious bulges on a V8 solid culture medium; the bacillus amyloliquefaciens BZ-29 can hardly grow on an OA solid medium. The image display of the bacillus amyloliquefaciens BZ-29 is shown in figure 2, the bacillus amyloliquefaciens BZ-29 presents bar shapes with different lengths under an electron microscope, more filiform structures are arranged among the thalli, and obvious bud tubes are arranged at the tail ends of the thalli. In the invention, the bacillus amyloliquefaciens BZ-29 can ferment glucose to produce acid and gas, can utilize citrate, can not ferment inositol and glycerol, has positive V-P test, has negative methyl red test, can not produce gelatinase, can not decompose urea, can produce catalase, can produce siderophores, can produce IAA, and has phosphate dissolving capacity.
The total 16S rDNA sequence of the bacillus amyloliquefaciens BZ-29 is 1396bp, and is shown as SEQ ID NO. 1.
SEQ ID NO.1：
ATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTACCTTTAGGAGCCAGCC。
The obtained 16S rDNA sequence of the bacillus amyloliquefaciens BZ-29 is subjected to BLAST analysis and comparison with sequences in a GenBank database, and a constructed molecular identification clustering map is shown in figure 3. Comparison of the NCBI database shows that the similarity between the 16S rDNA sequence of the bacillus amyloliquefaciens BZ-29 and Bacillus amyloliquefaciens is over 99 percent.
The invention provides the fermentation liquor of the bacillus amyloliquefaciens BZ-29.
The invention provides a preparation method of the fermentation broth, which comprises the following steps:
culturing the bacillus amyloliquefaciens BZ-29 in a culture medium to obtain fermentation broth.
The bacillus amyloliquefaciens BZ-29 is cultured in a culture medium to obtain fermentation broth. In the present invention, the medium is preferably an LB medium, a GS medium, a PDA medium or a V8 medium, more preferably an LB medium. The temperature of the culture according to the invention is preferably 25℃to 30℃and more preferably 28 ℃; the time of the culture is preferably 3 to 7 days, more preferably 5 days. In the present invention,the cultivation is preferably carried out in a shaking table, preferably at a rotation speed of 150 to 200rpm, more preferably 180rpm. In the present invention, the cultivation is preferably performed under dark conditions. The concentration of the fermentation broth obtained by the culture of the invention is preferably 1X 10 9 ～1×10 10 CFU/mL, more preferably 1X 10 10 CFU/mL。
The invention also provides application of the bacillus amyloliquefaciens BZ-29 or the fermentation broth in promoting plant growth and/or preventing and controlling diseases.
In the present invention, the plant preferably includes any one or two or more of Atractylodis rhizoma, corn and tobacco. In the present invention, the promotion of plant growth preferably includes any one or two or more of the following (1) to (4):
(1) Promoting the absorption of nutrient elements by plants;
(2) Promoting stem growth;
(3) Promote root elongation;
(4) The dry weight and fresh weight of the plants are improved.
In the present invention, the Bacillus amyloliquefaciens BZ-29 can promote plant growth, more preferably can promote plant seedling growth. In the present invention, the Bacillus amyloliquefaciens BZ-29 can promote the absorption of nutrient elements by plants, wherein the nutrient elements preferably comprise phosphorus. The bacillus amyloliquefaciens BZ-29 can promote the growth of plant stems, and the bacillus amyloliquefaciens BZ-29 can promote the increase of the stem length of bighead atractylodes rhizome by 91.67%; can promote the increase of the tobacco stem length by 287.5%; can promote the increase of corn stem length by 39.6 percent. The bacillus amyloliquefaciens BZ-29 can promote the elongation of plant roots, and the bacillus amyloliquefaciens BZ-29 can promote the increase of the root length of bighead atractylodes rhizome by 88.24%; can promote the increase of the root length of tobacco by 41.7 percent; can promote the increase of corn root length by 23.7 percent. The bacillus amyloliquefaciens BZ-29 provided by the invention can preferably improve the dry weight and fresh weight of plants, and the bacillus amyloliquefaciens BZ-29 provided by the invention can promote the increase of the fresh weight of bighead atractylodes rhizome by 175.99% and the dry weight by 132.50%; can promote the fresh weight of tobacco to increase by 291.5 percent and the dry weight to increase by 393.9 percent; can promote the fresh weight of corn to be increased by 42.6 percent and the dry weight to be increased by 6.3 percent.
In the invention, the bacillus amyloliquefaciens BZ-29 can prevent and treat diseases. The control of diseases according to the invention preferably comprises control of pathogenic microorganisms. In the invention, the bacillus amyloliquefaciens BZ-29 has antagonistic effect on various pathogenic fungi such as peanut southern blight germ, rice damping-off germ, wheat take-all germ, rice sheath blight germ, wheat stem rot germ, rape sclerotium germ and the like.
The invention also provides an application of the bacillus amyloliquefaciens BZ-29 or the zymotic fluid in promoting the synthesis of sesquiterpene lactones from bighead atractylodes rhizome. The promotion of the synthesis of the sesquiterpene lactone by the bighead atractylodes rhizome is preferably realized by having a remarkable promotion effect on the transformation of the key gene DXR, FPPS, HMGR of the sesquiterpene lactone by the white atractylodes rhizome.
The invention also provides a using method of the bacillus amyloliquefaciens BZ-29 or the fermentation broth, and the bacillus amyloliquefaciens BZ-29 or the fermentation broth is applied when the plant seeds are in a white period and/or the plants grow to 5-7 leaves; the application amount is 1-5 mL/plant.
In the present invention, the plant height is preferably 5 to 20cm, more preferably 15cm, when the plant is applied when growing to 5 to 7 leaves. The present invention is preferably applied near the seeds and/or plants when applied. The amount of the present invention to be applied is preferably 1 to 5 mL/plant, more preferably 1 mL/plant.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Isolation of strains
The endophytic bacteria BZ-29 of the bighead atractylodes rhizome is obtained from the healthy bighead atractylodes rhizome plant body in Yuexi county of Anhui province by adopting a flat plate coating method to separate, purify and screen.
The specific operation is as follows:
washing fresh rhizoma Atractylodis Macrocephalae rhizome with tap water to remove surface soil, removing fibrous root, cutting rhizome into square with side length of about 5mm in aseptic ultra-clean benchSterilizing the bulk sample block in 75% alcohol solution for 10min, and transferring to volume fraction of 30% H 2 O 2 Sterilizing the solution for 30s, washing 3 times with sterile water, and coating the plate with sterile water washed for the last time as a control; placing a sample in a sterile centrifuge tube, adding a proper amount of sterile water, adding 2 steel balls (with diameters of 2mm and 4mm respectively) with different sizes, vibrating for 2min at 60Hz, sucking 100 mu L of solution from the centrifuge tube onto an LB solid plate culture medium, uniformly coating by a sterile coater, sealing and storing in a dark constant temperature incubator at 28 ℃, observing in time, picking up a new solid culture medium plate after bacteria grow out, repeatedly operating on the LB solid culture medium by a scribing method until a single bacterial colony with stable growth is obtained, and storing in a 50% glycerol solution to obtain the strain BZ-29.
2. Strain Performance determination
(1) Determination of IAA secretion Capacity
The strain BZ-29 is streaked on LB solid medium and is first cultured for 24 hours under the dark condition at 28 ℃.
After the first culture is finished, single colonies are picked into NB liquid culture medium with 0.5g/L tryptophan, and the second culture is carried out for 72 hours under the dark condition of 28 ℃ and 180 r/min.
After the second culture is finished, 1mL of bacterial liquid is sucked, centrifugation is carried out for 10min at 10000r/min, supernatant is taken, IAA indicator with volume of about 2 times is added, shading and color development are carried out for 30min, whether red color is generated or not is observed, the absorbance value at the wavelength of 530nm of the color development solution is measured by using an ultraviolet spectrophotometer, and the IAA concentration in the bacterial liquid is calculated according to the drawn IAA standard curve.
The IAA standard curve is drawn by an IAA gradient dilution measuring method. Using IAA standards, 0.1. Mu.g/mL, 0.5. Mu.g/mL, 1.0. Mu.g/mL, 1.5. Mu.g/mL, 2.0. Mu.g/mL, 2.5. Mu.g/mL standards were prepared and assayed at OD 530nm The following absorbance values, IAA concentration and absorbance are shown in table 1, with IAA concentration as x-axis and absorbance as y-axis, the standard curve is shown in fig. 5, and the formula of the obtained standard curve is: y=0.0408x-0.003, r 2 ＝0.9836。
TABLE 1 IAA concentration and absorbance thereof
| IAA concentration (μg/mL) | Absorbance (OD value) |
| 0.1 | 0.001 |
| 0.5 | 0.016 |
| 1.0 | 0.034 |
| 1.5 | 0.064 |
| 2.0 | 0.084 |
| 2.5 | 0.093 |
As shown in FIG. 4, strain BZ-29 produced a clear red color with IAA-producing ability. And (3) checking the quantitative result to obtain a light absorption value of 0.343, and calculating the IAA concentration in the bacterial liquid according to the drawn IAA standard curve. The capability of producing IAA of BZ-29 can reach 8.48 mug/mL.
(2) Determination of phosphate-solubilizing ability
Qualitative detection: the whole single colony of the strain BZ-29 is picked on an NBRIP solid culture medium by using a 5mm puncher, cultured for 2-3D under a constant temperature incubator at 28 ℃ and a dark condition, whether transparent phosphate solubilizing rings are generated around the colony is observed, and the diameter D of the phosphate solubilizing rings, namely the phosphate solubilizing rings and the diameter D of the colony are measured.
Quantitative detection: determining phosphorus dissolving capacity of test strain by molybdenum-antimony colorimetric method, transferring seed solution of strain BZ-29 into NBRIP liquid culture medium with 1% inoculum size by volume percentage, culturing at 28deg.C under 180rpm in dark condition for 7d, centrifuging fermentation broth at 8000r/min for 10min, collecting supernatant 100 μl, adding distilled water to constant volume of 10mL, sequentially adding 0.1g/mL ascorbic acid solution and ammonium molybdate-sulfuric acid mixed solution 200 μl, shaking thoroughly, standing for 5min, and measuring OD by spectrophotometer 822nm The absorbance at the wavelength is zeroed before measurement.
By KH 2 PO 4 (baked to constant weight at 105 ℃ C. Before use) 1, 2, 3, 4, 5mg/L standard solution (in-situ preparation) was prepared, and the OD of the standard solution was measured by the above method 822nm The absorbance values, phosphorus content and absorbance values are shown in Table 2, and a phosphorus standard curve is drawn with the absorbance value as the x-axis and the phosphorus content as the y-axis. The standard curve is shown in fig. 6, and the formula of the obtained standard curve is: y= 75.718x-182.2, r 2 ＝0.9805。
Preparing an ammonium molybdate-sulfuric acid mixed solution: 0.14g/mL ammonium molybdate solution, 6.0mol/L H 2 SO 4 The solution and 0.03g/mL of potassium antimony tartrate solution are mixed according to the volume ratio of 9:40:1 (prepared in situ)
TABLE 2 phosphorus content and absorbance thereof
| Phosphorus content (mmol/L) | Absorbance value |
| 2 | 2.438 |
| 4 | 2.459 |
| 6 | 2.476 |
| 8 | 2.516 |
| 10 | 2.539 |
As shown in FIG. 7, a transparent phosphate solubilizing ring was formed around the colony, the diameter D of the phosphate solubilizing ring was 2.867.+ -. 0.058cm, and the diameter D of the colony was d=0.933.+ -. 0.153cm. And (5) checking the quantitative result to obtain a light absorption value of 48.200, and calculating the phosphorus content in the bacterial liquid according to the drawn phosphorus content standard curve. The phosphate dissolving capacity of the obtained strain BZ-29 can reach 3.47mol/L.
(3) Detection of cellulase production
The strain BZ-29 is cultivated on LB medium under dark condition at 28 ℃ for 24 hours at 180r/min until single colony is grown.
The whole single colony of the strain BZ-29 is picked on a hydroxycellulose nano solid culture medium by using a 5mm puncher, cultured for 3d under a constant temperature incubator at 28 ℃ and a dark condition, the culture medium is dyed for 30min by using Congo red solution, the culture medium is slowly washed by pure water and decolorized for 10min by using 1mol/L NaCl solution, and whether a pale yellow transparent ring appears around the growth of the strain BZ-29 is observed.
As a result, as shown in FIG. 8, a pale yellow transparent ring was observed around the growth of the strain BZ-29, indicating that the strain was able to produce cellulase.
(4) Detection of iron production carrier capacity
The strain BZ-29 is cultivated on LB medium under dark condition at 28 ℃ for 24 hours at 180r/min until single colony is grown.
The whole colony of the strain BZ-29 was picked up on a CAS solid medium by using a 5mm puncher, cultured for 1-2 d in a constant temperature incubator at 28℃under dark conditions, and observed whether yellow color was generated around the colony.
As a result, as shown in FIG. 9, yellow production was observed around the colonies, indicating that the strain BZ-29 had the ability to produce siderophores.
(5) Determination of antagonistic pathogenic fungi Capacity
The strain BZ-29 is cultivated on LB medium under dark condition, the cultivation temperature is 28 ℃, and the cultivation is carried out for 24 hours at 180r/min until single colony is grown.
The whole single colony of strain BZ-29 was picked up on PDA solid medium inoculated with pathogenic fungi using a 5mm punch as an experimental group. Meanwhile, a control group is set, and pathogenic fungi are inoculated only on a PDA culture medium, and the strain BZ-29 is not inoculated. The experimental group and the control group are respectively cultivated in a constant temperature incubator at 28 ℃ for 2-5 d under the dark condition, and when the control group grows full of the whole plate culture medium, the growth condition of pathogenic fungi of the experimental group is observed.
The bacterial strain BZ-29 has antagonistic effect on various pathogenic fungi such as peanut southern blight germ, rice damping-off germ, wheat take-all germ, rice sheath blight germ, wheat stem rot germ and sclerotium germ, wherein the antagonistic effect of the bacterial strain BZ-29 on the pathogenic fungi of peanut southern blight germ, rice damping-off germ, wheat take-all germ and rice sheath blight germ is shown in figure 10.
3. Identification of strains
(1) Colony morphology observation
BZ-29 strain is inoculated on LB, GS, PDA, V and OA solid culture media respectively by streaking, cultured for 16 hours under the dark condition at the temperature of 28 ℃, and the single colony morphology is observed and recorded.
Wherein the LB solid medium, the GS solid medium and the OA solid medium are purchased from Qingdao high-tech industrial Yuan Haibo biotechnology Co., ltd; PDA solid medium was purchased from Hangzhou Baisi biotechnology Co., ltd; v8, self-preparation: v8 vegetable juice (Campbell Soup Company) 100mL, caCO 3 (Shalongchemical Co., ltd.) 0.2g, and purified water was set to 1L. All the culture media are sterilized at 121 ℃ for 20min after being prepared.
The single colony is shown in figure 1, and the strain BZ-29 is light gray, thick on the surface, smooth or wrinkled and has bulges on LB solid medium; the GS solid culture medium is bright white, has thick and smooth surface and is provided with bulges; the PDA solid culture medium is gray white, has thick and smooth surface and obvious bulges; the V8 solid culture medium is light off-white, has a sticky surface, is smooth or is wrinkled, and has obvious bulges; little growth was possible on OA solid medium.
(2) Observation of the form of the fungus
Taking BZ-29 strain bacterial liquid which is cultured in an LB liquid culture medium for 24 hours, and centrifuging; repeatedly washing with sterile water, and centrifuging. Adding a proper amount of glutaraldehyde fixing solution (prepared by 0.1M phosphate buffer solution) with the volume fraction of 2.5% into the precipitated thalli, storing for 12 hours at 4 ℃, soaking and washing for 3 times by using 0.1M phosphate buffer solution, centrifuging to precipitate, absorbing supernatant, adding 30% ethanol water solution with the volume fraction, standing for 20 minutes at 4 ℃, centrifuging to precipitate, and then washing the precipitate sequentially by using 50% ethanol solution, 70% ethanol solution, 80% ethanol solution, 90% ethanol solution and 100% ethanol solution with the volume fraction, so as to dehydrate the thalli, wherein the specific washing step is the same as the washing step of 30% ethanol solution with the volume fraction; finally, the supernatant is sucked and removed, the mixture is replaced to 100 percent of acetone, and the mixture is kept stand for 20 minutes at the temperature of 4 ℃ and repeated twice; and sucking the thalli, drying the sample by using a critical point dryer, taking out the dried sample, and observing the dried sample in a scanning electron microscope.
The observation result of the scanning electron microscope is shown in fig. 2, the strain BZ-29 presents a bar shape with different lengths under the electron microscope, more filiform structures are arranged among the thalli, and the tail ends of the thalli are provided with obvious bud tubes.
(3) Physiological and biochemical characteristics
The physiological and biochemical characteristics of BZ-29 strain were determined by reference to the methods recommended in the eighth edition of the "Berger's Manual of bacteria identification". The test results are shown in Table 3.
TABLE 3 physiological and biochemical characteristics of strains
Note that: +: a positive reaction; -: negative reaction
The physiological and biochemical characteristic results are shown in table 3 and fig. 11, and the physiological and biochemical characteristic detection results show that the strain BZ-29 is gram-negative bacteria; glucose is fermented to produce acid and gas, inositol and glycerol cannot be fermented, the V-P test is positive, the methyl red test is negative, gelatinase cannot be produced, urea cannot be decomposed, and catalase can be produced.
(4) Molecular characterization
The DNA of strain BZ-29 was extracted by the usual CTAB method.
After DNA of the strain BZ-29 was extracted, PCR was performed.
PCR reaction system: 2X Rapid Taq Master Mix. Mu.L, 2. Mu.L each of Primer1 and Primer2, 5. Mu.L of Template DNA, add ddH 2 O to 50. Mu.L. RapidTaq MasterMix is available from Nanjinouzan Biotech Inc.
PCR reaction conditions: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s, annealing at 51℃for 15s, extension at 72℃for 30s, and cycling for 35 times; finally, the extension is carried out for 5min at 72 ℃.
The primer sequences used were: the sequence of Primer1 is 27F and is SEQ ID NO.2, and is 5'-AGAGTTTGATCCTGGCTCAG-3' specifically; 1492R has the sequence of SEQ ID NO.3, specifically 5'-TACGGCTACCTTGTTACGACTT-3'.
The sequencing was performed by sending the amplified PCR stock to Nanjing qing department Biotechnology Co.
The obtained strain 16S rDNA sequence is shown as SEQ ID NO.1 。 And (3) comparing the obtained SEQ ID NO.1 sequence with sequences in a GenBank database through BLAST analysis, and constructing a molecular identification cluster map as shown in figure 3.
As can be seen from FIG. 3, strain BZ-29 and Bacillus amyloliquefaciens are gathered in the same branch, and NCBI comparison shows that the similarity is more than 99%.
The BZ-29 strain is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) according to morphological characteristics, culture characteristics, physiological and biochemical characteristics and molecular biological identification results. Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BZ-29 strain is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC M20221991 in the year 2022, 12 and 19. Deposit center address: university of martial arts, post code: 430072.
example 2
The preparation method of the fermentation broth of the bacillus amyloliquefaciens BZ-29 comprises the following steps:
inoculating bacillus amyloliquefaciens BZ-29 into LB culture solution for culture, wherein the culture condition is 180rpm, and the shake culture fermentation is carried out at 28 ℃ for 5 days. The concentration of the fermentation broth obtained by culture was 1X 10 10 CFU/mL。
Application example 1
Effect of Strain BZ-29 on white atractylodes rhizome growth
Plant cultivation is carried out in a greenhouse environment at 22 ℃. Cleaning Atractylodis rhizoma seeds with clear water, sterilizing with 75% alcohol for 10min, soaking in warm water for 2 hr, taking out, washing with sterile water, broadcasting in seedling tray, spraying appropriate amount of clear water, covering soil for about 1cm, and covering with preservative film; after the seeds are exposed to white, the seeds with the same exposure degree are selected and randomly grouped, and the seeds are divided into a test group and a control group. Of these, test group 5 strains, control group 5 strains, 3 replicates were run per group. The experimental group added 1 mL/strain of the fermentation broth described in example 2 near the seeds that were exposed to the same extent. The control group was supplemented with 1 mL/strain of LB medium without inoculation. After the experimental group and the control group grow for 30d, the bighead atractylodes rhizome plants are taken out from the soil, the stem height, the root length, the dry weight and the fresh weight are measured and respectively averaged, and the growth condition of the plants is observed.
The dry weight treatment method is that plants are placed in a glass culture dish which is weighed in advance, the plants are dried in an oven at 80 ℃ for 5 days, the total weight is taken out, and the mass of the culture dish is subtracted to obtain the dry weight of a sample.
The growth of bighead atractylodes rhizome in the experimental group and the control group is shown in table 4, fig. 12 and fig. 13.
TABLE 4 growth conditions, mean of Atractylodis rhizoma of experimental and control groups
| Group of | Stem height (cm) | Root length (cm) | Fresh weight (mg) | Dry weight (mg) |
| Test group | 6.9 | 6.4 | 313.8 | 18.6 |
| Control group | 3.6 | 3.4 | 113.7 | 8.0 |
As can be seen from Table 4 and FIGS. 12 to 13, the Bacillus amyloliquefaciens BZ-29 provided by the invention can promote the growth of bighead atractylodes rhizome. The bacillus amyloliquefaciens BZ-29 can promote the increase of the stem length of bighead atractylodes rhizome by 91.67%, the increase of root length by 88.24%, the increase of fresh weight by 175.99% and the increase of dry weight by 132.50%.
Application example 2
Effect of Strain BZ-29 on tobacco growth
Plant cultivation is carried out in a greenhouse environment at 22 ℃. Cleaning tobacco seeds with clear water, sterilizing with 75% alcohol for 10min, soaking in warm water for 2h, taking out, washing with sterile water, broadcasting in seedling tray, spraying appropriate amount of clear water, covering soil for about 1cm, and covering with preservative film; after the seeds are exposed to white, the seeds with the same exposure degree are selected and randomly grouped, and the seeds are divided into a test group and a control group. Of these, test group 5 strains, control group 5 strains, 3 replicates were run per group. The experimental group added 1 mL/strain of the fermentation broth described in example 2 near the seeds that were exposed to the same extent. The control group was supplemented with 1 mL/strain of LB medium without inoculation. After the experimental group and the control group grow for 30d, the tobacco plants are taken out from the soil, the stem height, the root length, the dry weight and the fresh weight are measured and respectively averaged, and the growth condition of the plants is observed.
The tobacco growth of the experimental and control groups are shown in table 5 and fig. 14 and 15.
Table 5 Experimental and control tobacco growth, mean
| Group of | Stem height (cm) | Root length (cm) | Fresh weight (mg) | Dry weight (mg) |
| Test group | 9.3 | 5.44 | 1084.0 | 32.1 |
| Control group | 2.4 | 3.84 | 276.9 | 6.5 |
As can be seen from Table 5 and FIGS. 14 to 15, the Bacillus amyloliquefaciens BZ-29 provided by the invention can promote tobacco growth. The bacillus amyloliquefaciens BZ-29 can promote the increase of the tobacco stem length and stem height by 287.5%, the root length by 41.7%, the fresh weight by 291.5% and the dry weight by 393.9%.
Application example 3
Effect of Strain BZ-29 on maize growth
Plant cultivation is carried out in a greenhouse environment at 22 ℃. Cleaning corn seeds with clear water, sterilizing with 75% alcohol for 10min, soaking in warm water for 2h, taking out, washing with sterile water, broadcasting in a seedling tray, spraying appropriate amount of clear water, covering the surface with soil for about 1cm, and covering with preservative film; after the seeds are exposed to white, the seeds with the same exposure degree are selected and randomly grouped, and the seeds are divided into a test group and a control group. Of these, test group 5 strains, control group 5 strains, 3 replicates were run per group. The experimental group added 1 mL/strain of the fermentation broth described in example 2 near the seeds that were exposed to the same extent. The control group was supplemented with 1 mL/strain of LB medium without inoculation. After the experimental group and the control group grow for 15d, the corn plants are taken out from the soil, the stem height, the root length, the dry weight and the fresh weight are measured and respectively averaged, and the growth condition of the plants is observed.
The corn growth conditions of the experimental group and the control group are shown in Table 6 and FIGS. 16 to 17.
TABLE 6 corn growth conditions for experimental and control groups, mean
| Group of | Stem height (cm) | Root length (cm) | Fresh weight (mg) | Dry weight (mg) |
| Test group | 26.53 | 20.75 | 2241.7 | 216.9 |
| Control group | 19.00 | 16.78 | 1572.6 | 204 |
As can be seen from Table 6 and FIGS. 16 to 17, the Bacillus amyloliquefaciens BZ-29 provided by the invention can promote the growth of corns. The bacillus amyloliquefaciens BZ-29 can promote the growth of corn stems by 39.6%, root length by 23.7%, fresh weight by 42.6% and dry weight by 6.3%.
Application example 4
Effect of strain BZ-29 on the amount of variant of the synthetic sesquiterpene lactone key gene DXR, FPPS, HMGR.
Plant cultivation is carried out in a greenhouse environment at 22 ℃. Cleaning Atractylodis rhizoma seeds with clear water, sterilizing with 75% ethanol for 10min, soaking in warm water for 2 hr, taking out, washing with sterile water, broadcasting in seedling tray, spraying appropriate amount of clear water, covering with soil for about 1cm, and covering with preservative film; when the plants grow to 5 leaves, the bighead atractylodes rhizome with the plant height of about 15cm is randomly divided into two groups, namely an experimental group and a control group. Of these, test group 5 strains, control group 5 strains, 3 replicates were run per group. 1 mL/strain of the fermentation broth described in example 2 was added near the rhizosphere of Atractylodes macrocephala in the experimental group. The control group was added with 1 mL/strain of LB liquid medium without inoculation.
Randomly picking the top leaf at 0d, 3d, 7d, 12d respectively, usingRNA was extracted using Vazyme RC411 RNA extraction kit, monod MonScript TM RTIII All-in-One Mix with dsDNase reverse transcription of RNA into cDNA was performed as template DNA using Monad Monamp TM The chemoshs qPCR Mix was subjected to qPCR assay to quantify the relative expression of DXR, FPPS, HMGR, and specific primers are shown in table 7.
Vazyme RC411 RNA extraction kit was purchased from Nanjinovone Biotech Co., ltd; monad MonScript TM RTIII All-in-One Mix with dsDNase is available from Mona (Suzhou) Biotechnology Co., ltd; monad Monamp TM Chemoshs qPCR Mix was purchased from monad biotechnology limited.
TABLE 7 primers used in qPCR assay
The qPCR test results are shown in FIG. 18, wherein the expression levels of the genes in the experimental group are the results of subtracting the expression level of the control group, the relative expression level of DXR, FPPS, HMGR is obviously up-regulated on the 3 rd day of adding the Bacillus amyloliquefaciens BZ-29 fermentation broth, and then the up-regulating amplitude is slowly reduced, and the relative expression level of DXR, FPPS, HMGR is still obviously higher than that of the culture medium control group on the 15 th day. The strain BZ-29 has remarkable promotion effect on the transformation of the key gene DXR, FPPS, HMGR of the sesquiterpene lactone synthesized by the leukorrheal technique.
In conclusion, the bacillus amyloliquefaciens BZ-29 provided by the invention can promote growth of various plants such as bighead atractylodes rhizome, tobacco, corn and the like, and meanwhile, the strain BZ-29 has a remarkable promoting effect on transformation of a key gene DXR, FPPS, HMGR of sesquiterpene lactone synthesized by white atractylodes, and can promote synthesis of sesquiterpene lactone.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
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Claims (10)
1. The bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BZ-29 is characterized in that the preservation number of the bacillus amyloliquefaciens BZ-29 is CCTCC NO: M20221991.
2. The bacillus amyloliquefaciens BZ-29 of claim 1, wherein the 16S rDNA sequence of the bacillus amyloliquefaciens BZ-29 is shown in SEQ ID NO. 1.
3. The fermentation broth of bacillus amyloliquefaciens BZ-29 of claim 1 or 2.
4. A method for preparing a fermentation broth according to claim 3, comprising the steps of:
culturing the bacillus amyloliquefaciens BZ-29 in a culture medium to obtain fermentation broth.
5. The use of bacillus amyloliquefaciens BZ-29 according to claim 1 or 2, the fermentation broth according to claim 3 or the fermentation broth prepared by the preparation method according to claim 4 for promoting plant growth and/or preventing and controlling diseases.
6. The use according to claim 5, wherein the plant comprises any one or more of white atractylodes rhizome, corn and tobacco.
7. The use according to claim 5, wherein promoting plant growth comprises any one or two or more of the following (1) to (4):
(1) Promoting the absorption of nutrient elements by plants;
(2) Promoting stem growth;
(3) Promote root elongation;
(4) The dry weight and fresh weight of the plants are improved.
8. The use according to claim 5, wherein the disease control comprises control of plant diseases caused by one or more fungi selected from the group consisting of southern blight of peanut, rhizoctonia solani, wheat take-all, sheath blight of rice, stem rot of wheat and sclerotium rot of rape.
9. The application of bacillus amyloliquefaciens BZ-29 according to claim 1 or 2, the fermentation broth according to claim 3 or the fermentation broth prepared by the preparation method according to claim 4 in promoting the synthesis of sesquiterpene lactones from bighead atractylodes rhizome.
10. The bacillus amyloliquefaciens BZ-29 according to claim 1 or 2, the fermentation broth according to claim 3 or the fermentation broth prepared by the preparation method according to claim 4, wherein the application is carried out during the plant seed white period and/or when plants grow to 5-7 leaves; the application amount is 1-5 mL/plant.
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| CA2880276A1 (en) * | 2012-08-14 | 2014-02-20 | Marrone Bio Innovations, Inc. | Bacillus sp. strain with antifungal, antibacterial and growth promotion activity |
| CN103952332A (en) * | 2014-03-14 | 2014-07-30 | 南京博农生物科技有限公司 | Bacillus amyloliquefaciens ZH1 and its application |
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| CA2880276A1 (en) * | 2012-08-14 | 2014-02-20 | Marrone Bio Innovations, Inc. | Bacillus sp. strain with antifungal, antibacterial and growth promotion activity |
| CN102839142A (en) * | 2012-09-14 | 2012-12-26 | 杨凌绿都生物科技有限公司 | Bacillus amyloliquefaciens (Ba 168) and fermenting culture method and application of bacillus amyloliquefaciens |
| CN103952332A (en) * | 2014-03-14 | 2014-07-30 | 南京博农生物科技有限公司 | Bacillus amyloliquefaciens ZH1 and its application |
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