CN111117920B - Bacillus mycoides producing protease and siderophore and application thereof - Google Patents
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Abstract
The invention provides bacillus mycoides producing protease and siderophore and application thereof, belonging to the technical field of microorganisms. The strain is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 12 and 19 months, and the biological preservation number of the strain is CGMCC No. 19224. The bacillus mycoides obtained by screening can obviously improve the iron nutrition condition of ginger in the growth period, enhance the root activity of ginger, promote the growth of ginger ground diameter, improve plant biomass and increase the dry and wet weight of ginger stem leaves, thereby proving the growth promotion effect of the strain provided by the invention and effectively enriching the strain resources of plant growth promotion bacteria.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to bacillus mycoides producing protease and siderophores and application thereof.
Background
The information disclosed in this background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Ginger is not only a common traditional Chinese medicine raw material, but also an indispensable seasoning in daily life. With the increasing demand of the ginger, the planting area of the ginger is more and more, and the planting area of the ginger in China reaches 29.9 kilohm by 20182. With the popularization of planting, the soil-borne diseases of the ginger become increasingly serious; the long-term use of chemical pesticides and fertilizers leads to further aggravation of environmental pollution, damages to the structure and physicochemical properties of soil, and causes problems of nutrient imbalance, hardening and the like of the soil. Researches show that the biological agent can effectively inhibit the problems and improve the quality and the yield of agricultural products. Therefore, the screening of the ginger growth-promoting bacteria and the preparation of the biological agent lay a good foundation for green production.
At present, the discovered plant growth-promoting bacteria such as Bacillus (Bacillus), Brevibacillus (Brevibacillus), Paenibacillus (Paenibacillus), Acinetobacter (Acinetobacter), Agrobacterium (Agrobacterium), Streptomyces (Streptomyces) and the like show good growth-promoting effects on various crops such as cotton, wheat, tobacco, hot pepper, tomato, potato and the like. The researches on ginger biocontrol bacteria are more, and few reports are made on growth-promoting bacteria with the effects of promoting the growth of ginger and improving iron nutrition.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide the bacillus mycoides producing protease and siderophores and the application thereof.
In order to achieve the purpose, the invention discloses the following technical scheme:
the invention provides a Bacillus mycoides strain TA-203 which is preserved in China general microbiological culture Collection center (address: No. 3 Xilu No.1 of the sunward area of Beijing, China) in 19.12.2019, and the biological preservation number of the strain is CGMCC No. 19224.
In a second aspect of the present invention, there is provided a microbial inoculant comprising said Bacillus mycoides TA-203. Experiments prove that the microbial inoculant can remarkably promote the growth of ginger after the ginger is subjected to root irrigation treatment after seedling setting.
Specifically, the microbial inoculant is obtained by inoculating the Bacillus mycoides (Bacillus mycoides) TA-203 into a liquid culture medium for culture;
wherein, the liquid culture medium is preferably LB culture medium;
the LB culture medium comprises the following components: yeast extract, 0.5%, peptone, 1%, sodium chloride, 1%, distilled water, 1000 mL. Sterilizing at 121 deg.C for 20min, and pH 7.0.
The culture conditions are specifically as follows: culturing for 20-30 h (preferably 24h) at 30-40 ℃ (preferably 37 ℃), rotating speed: 160-200rpm (preferably 180 rpm).
In a third aspect of the invention, there is provided the use of Bacillus mycoides TA-203 and/or microbial inoculant as described above in any one or more of a) to d) below:
a) producing protease;
b) producing iron carriers;
c) improving the nutrition condition of the plant rhizosphere soil;
d) promoting the growth of plants.
The plant is specifically ginger, and the improvement on the rhizosphere soil nutrition of the plant is specifically that the bacillus mycoides TA-203 and/or the microbial inoculant can generate protease and siderophores in soil, so that the rhizosphere soil nutrition of the ginger is improved.
The specific expression of promoting plant growth is as follows: improving iron nutrition status of rhizoma Zingiberis recens during growth period, enhancing root system activity of rhizoma Zingiberis recens, promoting growth of ground diameter of rhizoma Zingiberis recens, and increasing dry and wet weight of stem and leaf of rhizoma Zingiberis recens.
In a fourth aspect of the present invention, there is provided a method for promoting the growth of ginger, which comprises applying the above-mentioned bacillus mycoides TA-203 and/or microbial inoculant to rhizosphere soil of ginger plants after ginger seedling setting. Specifically, the application mode includes but is not limited to spraying and pouring; more specifically, the administration mode is that the bacillus mycoides TA-203 and/or the microorganism inoculant is subjected to root irrigation treatment.
Compared with the prior art, the invention has the following beneficial effects:
the invention reports a bacillus mycoides strain producing protease and siderophore for the first time, and a pot experiment proves that the bacterial liquid of the bacillus mycoides strain producing protease and siderophore can be used for root irrigation treatment after ginger is subjected to final singling, so that the iron nutrition condition in the ginger growth period can be effectively improved, the root activity of ginger can be enhanced, the growth of ginger ground diameter can be promoted, the plant biomass can be improved, and the dry and wet weight of ginger stem leaves can be increased. The bacillus mycoides of the invention has good growth promoting effect on ginger, thus having good value of practical application.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a microscopic examination of Bacillus mycoides in example 1 of the present invention.
FIG. 2 is a colony diagram of LB plate of Bacillus mycoides in example 1 of the present invention.
FIG. 3 is a diagram showing protease production ability of Bacillus mycoides in example 2 of the present invention.
FIG. 4 is a diagram showing the ability to produce siderophores of Bacillus mycoides in example 2 of the present invention.
FIG. 5 is a diagram showing the growth promoting effect of the Bacillus mycoides potting test in example 3 of the present invention.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The present invention will now be further described with reference to specific examples, which are provided for the purpose of illustration only and are not intended to be limiting. If the experimental conditions not specified in the examples are specified, the conditions are generally as usual or as recommended by the reagents company; reagents, consumables and the like used in the following examples are commercially available unless otherwise specified.
In one embodiment of the invention, the invention provides a Bacillus mycoides strain TA-203 which is preserved in China general microbiological culture Collection center (address: No. 3 Xilu 1 Beijing Shanggang district, Beijing, China) in 19.12.2019, and the biological preservation number is CGMCC No. 19224.
The strain is determined to be Bacillus mycoides (bacillusmycoides) by determining the 16S rDNA gene sequence (shown in SEQ ID NO.1) of the strain and determining morphological characteristics and physiological and biochemical indexes.
Specifically, after the Bacillus mycoides (Bacillus mycoides) TA-203 is cultured on an LB plate culture medium (yeast extract, 0.5 percent, peptone, 1 percent, sodium chloride, 1 percent, agar, 1.6 percent, distilled water, 1000mL, sterilization at 121 ℃ for 20min and pH7.0) for 24 hours, the colony is in a circular regular shape, milk white, convex and opaque; the thallus is long-rod shaped, produces spores and is gram-positive.
The physiological and biochemical characteristics of the Bacillus mycoides TA-203 are as follows: positive starch hydrolysis test, positive gelatin liquefaction test, positive glucose fermentation test, negative arabinose fermentation test, positive mannitol fermentation test and positive xylose fermentation test.
In another embodiment of the present invention, there is provided a method for culturing Bacillus mycoides TA-203, specifically comprising: the activated Bacillus mycoides TA-203 is cultured in LB liquid medium under aerobic condition of 37 deg.C for 24 hr, such as shaking table (180 rpm).
In still another embodiment of the present invention, there is provided a microbial inoculant comprising said Bacillus mycoides TA-203.
In another embodiment of the present invention, the microbial inoculant can be in the form of a liquid, an emulsion, a suspension, a powder, a granule, a wettable powder or a water dispersible granule.
In another embodiment of the present invention, the microbial inoculant further comprises an adjuvant acceptable for microbial inoculum.
In yet another embodiment of the present invention, the adjuvant is selected from one or more of a dispersing agent, a wetting agent, a disintegrating agent, a binder, a defoaming agent, an anti-freezing agent, a thickener, a filler and a solvent. The invention has no special limitation on the sources and the like of the auxiliary materials acceptable by the microbial inoculum, and generally adopts the products sold in the market.
The dispersing agent is an anionic dispersing agent and/or a nonionic dispersing agent, and can be selected from one or more of sodium lignosulfonate, sodium naphthalene sulfonate formaldehyde condensate, sodium methylene dinaphthalene sulfonate, formaldehyde condensate sulfate, polycarboxylate, alkylphenol polyoxyethylene phosphate and fatty acid polyoxyethylene ester.
The humectant can be one or more selected from sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, fructus Gleditsiae Abnormalis powder, fructus Sapindi Mukouossi powder, tea seed cake powder and nekal BX.
The disintegrant may be selected from one or more of bentonite, ammonium sulfate, aluminum chloride, urea, magnesium chloride and glucose.
The binder may be selected from one or more of starch, diatomaceous earth, cyclodextrin, rosin, carboxymethyl cellulose, carboxyethyl cellulose, and carboxymethyl cellulose salts.
The defoaming agent can be one or more of C8-C20 fatty alcohol compounds, C10-C20 saturated fatty acid compounds, epoxidized soybean oil, ethanol, silicone compounds and organic silicone oil.
The antifreeze agent can be selected from one or more of sorbitol, ethylene glycol, polyethylene glycol, propylene glycol, glycerol, urea and sodium chloride.
The thickener may be selected from one or more of gelatin, xanthan gum, polyethylene glycol and polyvinyl alcohol.
The filler can be one or more of light calcium carbonate, diatomite, bentonite, attapulgite and white carbon black.
The solvent may be selected from water (preferably deionized water) or methyl oleate.
In another embodiment of the present invention, the use of the above-mentioned Bacillus mycoides TA-203 and/or microbial inoculant in whole or in part in a) to d) below is also within the scope of the present invention:
a) producing protease;
b) producing iron carriers;
c) improving the nutrition condition of the plant rhizosphere soil;
d) promoting the growth of plants.
The plant is specifically ginger, and the improvement of the rhizosphere soil nutrition of the plant is specifically that protease and siderophores are generated in soil by the bacillus mycoides TA-203 and/or the microbial inoculant, so that the rhizosphere soil nutrition of the ginger is improved.
The specific expression of promoting plant growth is as follows: improving iron nutrition status of rhizoma Zingiberis recens during growth period, enhancing root system activity of rhizoma Zingiberis recens, promoting growth of ground diameter of rhizoma Zingiberis recens, and increasing one or more of dry weight and wet weight of stem and leaf of rhizoma Zingiberis recens.
In another embodiment of the present invention, there is provided a method for promoting ginger growth, comprising applying the above-mentioned bacillus mycoides TA-203 and/or microbial inoculant to rhizosphere soil of ginger plants after ginger seedling setting. Modes of application include, but are not limited to, spraying, pouring. More specifically, the administration mode is that the bacillus mycoides TA-203 and/or the microorganism inoculant is subjected to root irrigation treatment.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are test methods in which specific conditions are indicated, and are generally carried out under conventional conditions.
Example 1: identification of strains
The bacterial strain which is obtained by screening and has the function of promoting the growth of the ginger is identified by 16S rDNA and physiological and biochemical indexes, and the bacterial strain is determined to be Bacillus mycoides. As shown in FIGS. 1 and 2, the colony and thallus characteristics of the Bacillus mycoides TA-203 are as follows: after 24 hours of culture on an LB culture medium, the colony is in a round regular shape, is milky white, convex and opaque; the thallus is long-rod shaped, produces spores and is gram-positive.
The physiological and biochemical characteristics of the strain are as follows: positive starch hydrolysis test, positive gelatin liquefaction test, positive glucose fermentation test, negative arabinose fermentation test, positive mannitol fermentation test and positive xylose fermentation test.
The 16S rDNA sequence of the Bacillus mycoides TA-203 is as follows:
AGGGCCTGCGGGGTGCTATACATGCAAGTCGAGCGAATGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACTGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGAAAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTTGGAGCCAGCCGCCTAAGGGGACCAT(SEQ ID No.1)。
example 2: growth promotion test of Bacillus mycoides TA-203 on ginger
The preparation method of the bacterial liquid comprises the following steps: transferring the strain preserved on the inclined plane into a test tube filled with 5mL of LB liquid medium, and putting the test tube into a constant temperature shaking table for culturing for 12h, wherein the temperature is as follows: 37 ℃, rotation speed: 180 rpm; inoculating the activated bacterial liquid into 50mL of LB liquid culture medium according to the inoculation amount of 1%, and putting the LB liquid culture medium into a shaking table for 24h, wherein the temperature is as follows: 37 ℃, rotation speed: and (4) preparing a bacterial liquid at 180 rpm.
Wherein, LB liquid culture medium: yeast extract, 0.5%, peptone, 1%, sodium chloride, 1%, distilled water, 1000 mL. Sterilizing at 121 deg.C for 20min, and pH 7.0.
Transplanting ginger seedlings into potting soil, diluting 2mL of bacterial liquid (dilution multiple is 100 times) after ginger seedling setting, and then performing root irrigation on each ginger plant in the potting by using 200mL of diluted bacterial liquid (the CK group is to perform root irrigation after 2mL of sterilized LB liquid culture medium is diluted to 200 mL). The growth promoting effect on ginger after 50 days of culture was examined, and the results are shown in table 1 and fig. 3. It can be seen that: under the condition of potting, after the root irrigation treatment is carried out on the ginger in the initial growth stage under the actual soil condition by using the liquid of the Bacillus mycoides TA-203, the ginger can generate a good growth promoting effect, the height of ginger plants, the ground diameter of the ginger, the maximum root length and the wet weight of stem leaves are greatly improved compared with those of a control group, and the remarkable or extremely remarkable difference is achieved.
TABLE 1 growth promoting Effect
Example 3: protease and siderophore production test on strain TA-203
The protease-producing and siderophore-producing abilities of Bacillus mycoides TA-203 were examined, and the results are shown in tables 2-3 and FIGS. 4-5.
TABLE 2 protease production results
TABLE 3 results of quantification of siderophores
In conclusion, the bacillus mycoides obtained by screening has the functions of producing protease, siderophores and the like, and meanwhile, the experimental method adopted by the screened strain for researching the growth promotion effect of the ginger under the potting condition is closer to the actual application state, and the effect on the growth promotion of the ginger in the production and planting is very obvious by directly using the soil planted ginger as the experimental sample and being close to the agricultural planting mode of the ginger. Meanwhile, the plant height, the maximum root length, the ground diameter, the wet weight of stems and leaves and the like of the potted ginger are detected, and the growth promoting effect of the strain on the ginger is effectively proved.
It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.
SEQUENCE LISTING
<110> Shandong university of agriculture
<120> Bacillus mycoides producing protease and siderophore and application thereof
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1455
<212> DNA
<213> Bacillus mycoides TA-20316S rDNA
<400> 1
agggcctgcg gggtgctata catgcaagtc gagcgaatgg attaagagct tgctcttatg 60
aagttagcgg cggacgggtg agtaacacgt gggtaacctg cccataagac tgggataact 120
ccgggaaacc ggggctaata ccggataaca ttttgaactg catggttcga aattgaaagg 180
cggcttcggc tgtcacttat ggatggaccc gcgtcgcatt agctagttgg tgaggtaacg 240
gctcaccaag gcaacgatgc gtagccgacc tgagagggtg atcggccaca ctgggactga 300
gacacggccc agactcctac gggaggcagc agtagggaat cttccgcaat ggacgaaagt 360
ctgacggagc aacgccgcgt gagtgatgaa ggctttcggg tcgtaaaact ctgttgttag 420
ggaagaacaa gtgctagttg aataagctgg caccttgacg gtacctaacc agaaagccac 480
ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttat ccggaattat 540
tgggcgtaaa gcgcgcgcag gtggtttctt aagtctgatg tgaaagccca cggctcaacc 600
gtggagggtc attggaaact gggagacttg agtgcagaag aggaaagtgg aattccatgt 660
gtagcggtga aatgcgtaga gatatggagg aacaccagtg gcgaaggcga ctttctggtc 720
tgtaactgac actgaggcgc gaaagcgtgg ggagcaaaca ggattagata ccctggtagt 780
ccacgccgta aacgatgagt gctaagtgtt agagggtttc cgccctttag tgctgaagtt 840
aacgcattaa gcactccgcc tggggagtac ggccgcaagg ctgaaactca aaggaattga 900
cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg aagaacctta 960
ccaggtcttg acatcctctg aaaaccctag agatagggct tctccttcgg gagcagagtg 1020
acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1080
gagcgcaacc cttgatctta gttgccatca ttaagttggg cactctaagg tgactgccgg 1140
tgacaaaccg gaggaaggtg gggatgacgt caaatcatca tgccccttat gacctgggct 1200
acacacgtgc tacaatggac ggtacaaaga gctgcaagac cgcgaggtgg agctaatctc 1260
ataaaaccgt tctcagttcg gattgtaggc tgcaactcgc ctacatgaag ctggaatcgc 1320
tagtaatcgc ggatcagcat gccgcggtga atacgttccc gggccttgta cacaccgccc 1380
gtcacaccac gagagtttgt aacacccgaa gtcggtgggg taaccttttt ggagccagcc 1440
gcctaagggg accat 1455
Claims (13)
1. Bacillus mycoides (A)Bacillus mycoides) TA-203, which is preserved in China general microbiological culture Collection center in 19 th 12 th 2019 with the biological preservation number of CGMCC No. 19224.
2. A microbial inoculant comprising the Bacillus mycoides of claim 1 (I), (II)Bacillus mycoides)TA-203。
3. The microbial inoculant according to claim 2, wherein the microbial inoculant is obtained by inoculating Bacillus mycoides TA-203 in a liquid medium for culturing.
4. A microbial inoculant according to claim 3 wherein the liquid medium is LB medium.
5. A microbial inoculant according to claim 3 wherein the fermentation conditions are in particular: culturing at 30-40 ℃ for 20-30 h, rotating speed: 160-200 rpm.
6. A microbial inoculant according to claim 5 wherein the fermentation conditions are in particular: incubation at 37 ℃ for 24h, rotation speed: 180 rpm.
7. The microbial inoculant according to claim 2 further comprising an adjuvant acceptable to microbial inoculants.
8. The microbial inoculant according to claim 7 wherein the adjuvants are selected from one or more of dispersants, wetting agents, disintegrants, binders, defoamers, antifreeze agents, thickeners, fillers and solvents.
9. The microbial inoculant according to claim 2, wherein the microbial inoculant is in the form of a liquid, an emulsion, a suspension, a powder, a granule, a wettable powder or a water dispersible granule.
10. Use of Bacillus mycoides TA-203 according to claim 1 and/or a microbial inoculant according to any one of claims 2 to 7 in whole or in part in a) to d) as follows:
a) producing protease;
b) producing iron carriers;
c) promoting the growth of ginger.
11. The use of claim 10, wherein promoting ginger growth comprises:
improving iron nutrition status of rhizoma Zingiberis recens during growth period, enhancing root system activity of rhizoma Zingiberis recens, promoting growth of rhizoma Zingiberis recens ground diameter, increasing plant biomass, and increasing one or more of dry weight and wet weight of stem and leaf of rhizoma Zingiberis recens.
12. A method for promoting the growth of ginger, comprising applying to rhizosphere soil of ginger plants a bacillus mycoides TA-203 according to claim 1 and/or a microbial inoculant according to any one of claims 2 to 9 after ginger seedling setting.
13. The method of promoting ginger growth according to claim 12, wherein the applying comprises: the ginger is subjected to root irrigation treatment by using the bacillus mycoides TA-203 and/or the microbial inoculant.
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