CN106434436A - Application of bacillus megatherium 1.1123 in preparation of microbial agent or fertilizer - Google Patents
Application of bacillus megatherium 1.1123 in preparation of microbial agent or fertilizer Download PDFInfo
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- CN106434436A CN106434436A CN201610824506.3A CN201610824506A CN106434436A CN 106434436 A CN106434436 A CN 106434436A CN 201610824506 A CN201610824506 A CN 201610824506A CN 106434436 A CN106434436 A CN 106434436A
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- bacillus megaterium
- phosphorus
- bacillus megatherium
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/11—Bacillus megaterium
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
Abstract
The invention discloses application of bacillus megatherium 1.1123 in preparation of a microbial agent or fertilizer. The bacillus megatherium 1.1123 is preserved in Microbial Culture Collection Center of Guangdong Institute of Microbiology (GIMCC) on August 30, 2016, and the preservation number is GIMCC 1.1123. The bacillus megatherium 1.1123 is a phosphate solubilizing microorganism and can convert difficultly-soluble or insoluble phosphorus into soluble phosphorus capable of being absorbed by plants, increase the content of plant-available effective phosphorus in soil and promote plant growth. Through measuring, the phosphate solubilizing capacity of the bacillus megatherium 1.1123 can reach 61.2%, the live bacterium number of a flask shaking fermentation test reaches 5.6*10<9>/mL, and the spore rate reaches 87.5%. Accordingly, the bacillus megatherium 1.1123 can be applied to microbial fertilizer production by serving as the agricultural microbial agent and has an important research and application value on agricultural planting.
Description
Technical field：
The invention belongs to field of agricultural microbial technology is and in particular to a kind of bacillus megaterium (Bacillus
Megaterium) 1.1123 in degraded slightly solubility or insoluble phosphorus, prepares the application in microbial bacterial agent or microbial-bacterial fertilizer.
Background technology：
During growth and development of plants, phosphorus plays an important role, and the yield of crop and quality are close with phosphorus utilization
Cut is closed.However, because being widely present of solidification phosphorus makes most of soil there is the situation lacking phosphorus, therefore, scarce phosphorus is restriction
One of most important factor in agricultural production both at home and abroad.From the point of view of domestic agriculture production modl, the whole nation has 74% arable soil to lack
Phosphorus, and in soil to be solved generally existing scarce phosphorus present situation, mainly can be realized by two kinds of approach, first is to increase phosphate fertilizer
Amount of application, second is to increase the utilization rate solidifying phosphorus in soil.Iff the input of the increase phosphate fertilizer single by approach one,
To solve the scarce phosphorus present situation in soil, both not met the sustainable development of agricultural, nor effectively, thoroughly to solve soil to lack
Phosphorus problem.Therefore, the utilization rate improving solidification phosphorus in soil had both met the developing direction of modern agriculture, was the high production of low input
Sustainable development, can effectively solve the problem that again and lacks phosphorus phenomenon in soil.
Improve the phosphate fertilizer utilization rate in soil, can be realized by following three kinds of modes, first is to inhale phosphorus according to crops
Characteristic, the P form distribution situation in research soil, improves the utilization rate of phosphorus by improving the modes such as the application technique of phosphate fertilizer；The
Two is the innovation by breeding technique, plays the intrinsic advantage of plant, and the kind that phosphorus efficiency utilizes, exploitation profit are screened in directive breeding
With insoluble phosphorus resource in soil；3rd is screening efficient phosphate-solubilizing microorganism, is produced by industrial fermentation, makes phosphorus decomposing microbial inoculum
Dress seed or be directly manured into soil, insoluble phosphorus in activating soil.
Bacillus megaterium (Bacillus megaterium) is as conduct in microbial manure bio-safety layered directory
One-level bacterial strain, has the effect of degraded slightly solubility or insoluble phosphorus, is widely used as the conventional bacterial strain of microbial manure.Mesh
Before, domestic manufacturer produces bacillus megaterium product generally existing and (formation gemma rate is low, and the preservation time is short) of low quality, bacterium
Strain functional deterioration and the problems such as phosphorus decomposing effect on driving birds is not good, the serious extensive application that govern this bacterial strain.
Content of the invention：
First purpose of the present invention is to provide bacillus megaterium (Bacillus megaterium) 1.1123 in degraded
Application in slightly solubility or insoluble phosphorus, described bacillus megaterium (Bacillus megaterium) 1.1123, this bacterium in
August is preserved in Guangdong Microbes Inst DSMZ, address on 30th within 2016：Guangzhou, Guangdong Yuexiu District martyr
Road 100 No. 59 building of compound, deposit number GIMCC 1.1123.
The biological property of bacillus megaterium (Bacillus megaterium) 1.1123 is as follows：
1st, strain morphology feature：After this bacterium is cultivated 24 hours on NA culture medium flat plate, bacterium colony is white, circular, surface
Smooth opaque, edge is incised；Gram's staining is the positive, and thalline is shaft-like, has the raw flagellum in end, motion, no presss from both sides film, in gemma
Raw, oval.
2nd, physiological and biochemical property：Bacillus megaterium (Bacillus megaterium) the 1.1123V-P test of the present invention
For feminine gender；Indole test is feminine gender；Nitrate reduction negative；Citrate is using negative；The glucose that carbon source produces in acid is sun
Property；Can hydrolyzed casein, urea, aesculin, beta galactose glycosides (ONPG)；20～45 DEG C of growth temperature range, optimal pH be 7.0～
7.5.
The chemotaxonomy status of bacillus megaterium (Bacillus megaterium) 1.1123：Extract huge gemma
The genomic DNA of bacillus (Bacillus megaterium) 1.1123 is simultaneously sequenced to its 16S rDNA, its nucleotide sequence
As SEQ ID NO:Shown in 1.This sequence is carried out BLAST comparative analysis with the known array in GenBank database, and from number
Obtain the 16S rDNA sequence of related species, phylogenetic tree construction according to storehouse.Phylogenetic tree shows this bacterial strain and huge gemma
Bacillus (Bacillus megaterium) has nearest similarity, is named as bacillus megaterium (Bacillus
megaterium)1.1123.
Second object of the present invention is to provide above-mentioned bacillus megaterium (Bacillus megaterium) 1.1123
Application in preparing microbial bacterial agent or microbial-bacterial fertilizer.
Third object of the present invention is to provide a kind of microbial bacterial agent it is characterised in that with above-mentioned huge gemma bar
Bacterium (Bacillus megaterium) 1.1123 is as active ingredient.
Fourth object of the present invention is to provide a kind of microbial-bacterial fertilizer it is characterised in that with above-mentioned huge gemma bar
Bacterium (Bacillus megaterium) 1.1123 is as active ingredient.
The bacillus megaterium (Bacillus megaterium) 1.1123 of the present invention is a kind of phosphate solubilizing microorganism, can
Slightly solubility or insoluble phosphorus are converted into titanium pigment, improve the available available phosphorus content of plant in soil, promote plant life
Long, reduce the use of chemical fertilizer.After measured, the dissolving P capacity of this bacterium reaches 61.2%, and shake flask fermentation test viable count reaches 5.6 ×
109Individual/mL, gemma rate reaches 87.5%.Therefore, the production of microbial-bacterial fertilizer can be applied to as agricultural microbial agent, right
Agricultural planting aspect has important research and using value.
The bacillus megaterium (Bacillus megaterium) 1.1123 of the present invention is preserved in August in 2016 on the 30th
Guangdong Microbes Inst DSMZ, address：Xianlie Middle Rd., Yuexiu Zone, Guangzhou, Guangdong 100 compound 59
Building, deposit number is：GIMCC 1.1123.
Brief description：
Fig. 1 is the transmission electron microscope photo (6000 of bacillus megaterium (Bacillus megaterium) 1.1123
Times)；
Fig. 2 is that bacillus megaterium (Bacillus megaterium) 1.1123 is according to 16S rDNA sequence construct
System develops tree；
Fig. 3 is the design sketch that bacillus megaterium (Bacillus megaterium) 1.1123 hydrolyzes organophosphor.
Specific embodiment：
Following examples are that the present invention is further illustrated, rather than limitation of the present invention.Method therefor and technology,
If no special instructions, it is conventional method and technology.
Embodiment 1：The biological character of bacillus megaterium (Bacillus megaterium) 1.1123 and Physiology and biochemistry
And Molecular Identification
S1.1 morphological feature
After bacillus megaterium (Bacillus megaterium) 1.1123 is cultivated 24 hours on NA culture medium flat plate,
Bacteria colony white, circular, smooth surface is opaque, and edge is incised；Gram's staining is the positive；Thalline is shaft-like, has the raw flagellum in end,
Motion, no presss from both sides film, raw in gemma, oval (Fig. 1 is shown in by transmission electron microscope photo).
S1.2 physiological and biochemical property
Through physiological and biochemical test, bacillus megaterium (Bacillus megaterium) the 1.1123V-P test of the present invention
For feminine gender；Indole test is feminine gender；Nitrate reduction negative；Citrate is using negative；The glucose that carbon source produces in acid is sun
Property；Energy hydrolyzed casein, urea, aesculin and beta galactose glycosides (ONPG)；20～45 DEG C of growth temperature range, optimal pH is 7.0
～7.5, the results are shown in Table 1.
Table 1 bacillus megaterium 1.1123 physiological and biochemical property
Note：+, positive；-, negative；WH, white；R is shaft-like
The sequence and analysis of the 16S rDNA of S1.3 bacillus megaterium (Bacillus megaterium) 1.1123
The quick preparation of S1.3.1DNA template：Bacillus megaterium (Bacillus megaterium) 1.1123 is rule
It is seeded on NA culture medium flat plate, cultivate 24 hours for 37 DEG C.Single bacterium colony is taken to be suspended in 10 μ L sterile distilled waters, in boiling water
Heating 5min, centrifugation, take supernatant as DNA profiling.
S1.3.2 16S rDNA gene PCR expands
Primer：Primer A：5’-AGAGTTTGATCCTGGCTCAG-3’；Primer B：5’-
AAGGAGGTGATCCACCCCCA-3’.
PCR reaction system is as shown in table 2：
Table 2PCR reaction system
| 10 × PCR buffer solution | 5μL |
| 2mmol/L dNTP | 4μL |
| 10pmol/L Primer A | 1μL |
| 10pmol/L Primer B | 1μL |
| 2U/L Tap enzyme | 0.4μL |
| DNA profiling | 1μL |
| Sterilizing ddH2O | 37.4μL |
| Cumulative volume | 50μL |
PCR amplification condition：94℃5min；94 DEG C of 1min, 56 DEG C of 1min, 72 DEG C of 2min, 30 circulations；72 DEG C, 7min.
S1.3.3 sequencing：PCR primer after purification, through sequencing, its nucleotide sequence such as SEQ ID NO:Shown in 1.Should
Sequence carries out BLAST comparative analysis with the known array in GenBank database, and obtains the 16S of related species from database
RDNA sequence, phylogenetic tree construction, as shown in Figure 2.Phylogenetic tree shows this bacterial strain and bacillus megaterium (Bacillus
Megaterium) there is nearest similarity, be named as bacillus megaterium (Bacillus megaterium) 1.1123.
Embodiment 2：Bacillus megaterium (Bacillus megaterium) 1.1123 hydrolysis organophosphor test
Organophosphor culture medium prescription：Glucose 10g, ammonium sulfate 0.5g, yeast extract 0.5g, sodium chloride 0.3g, potassium chloride
0.3g, magnesium sulfate 0.3g, ferrous sulfate 0.3g, manganese sulfate 0.03g, lecithin 0.2g, calcium carbonate 1.0g, agar 15g, water
1000mL, pH 7.0～7.5.
The organophosphor having sterilized culture medium is cooled to 45 DEG C about start to be down flat plate, is inverted in after flat board cooled and solidified
Place 24 hours in 37 DEG C of constant incubators it is therefore an objective to draining media surface moisture and verifying that flat board does not have pollution microbes.Will
1.1123 points of bacillus megaterium (Bacillus megaterium) is connected on drying and aseptic organophosphor culture medium flat plate,
Upside down is cultivated 7 days in 37 DEG C of incubators, observes periphery of bacterial colonies hydrolysis circle situation (as shown in Figure 3).As can be known from Fig. 3,
Bacillus megaterium (Bacillus megaterium) 1.1123 periphery of bacterial colonies define hydrolysis circle, illustrate that the present invention's is huge
Bacillus (Bacillus megaterium) 1.1123 has strong organophosphor hydrolysing activity.
Embodiment 3：Bacillus megaterium (Bacillus megaterium) 1.1123 hydrolysis organophosphor ability measures
Organophosphor Liquid Culture based formulas：Glucose 10g, ammonium sulfate 0.5g, yeast extract 0.5g, sodium chloride 0.3g, chlorine
Change potassium 0.3g, magnesium sulfate 0.3g, ferrous sulfate 0.3g, manganese sulfate 0.03g, lecithin 0.2g, calcium carbonate 1.0g, water 1000mL,
pH 7.0.
The preparation of seed liquor：150mL triangular flask fills organophosphor fluid nutrient medium 50mL, sterilizing.Picking one ring activates
Bacillus megaterium (Bacillus megaterium) 1.1123 thalline are inoculated in organophosphor fluid nutrient medium, 180rpm, and 37
DEG C culture 16h obtain seed liquor.
Liquid Culture：Prepare organophosphor fluid nutrient medium, liquid amount is 100mL/500mL conical flask.Seed by preparation
Liquid takes 5mL to access in organophosphor fluid nutrient medium respectively, and in 37 DEG C, 180rpm cultivates 7 days, and in triplicate, comparison does not connect bacterium, with
Sample is in triplicate.
Phosphorus content Specification Curve of Increasing：Accurately draw phosphorus standard liquid 0,0.2,0.4,0.6,0.8,1.0,1.2 and 1.4mL
(being equivalent to phosphorous 0,2,4,6,8,10,12 and 14 μ g), is respectively placed in 10mL tool plug test tube, adds molybdenum blue colorimetric mixed liquor
5mL, adds water to scale, mixing, after 45 DEG C of water-bath 25min, is made blank with No. 0 pipe, using visible spectrophotometer in 660nm
Mensuration absorbance at wavelength, draws phosphorus calibration curve with the absorbance measured to phosphorus content.
The mensure of phosphorus content in nutrient solution：Nutrient solution 5000rpm is centrifuged 10min, accurately draws 5mL supernatant and be placed in
In 10mL tool plug test tube, add 5mL colorimetric mixed liquor, in 45 DEG C of water-bath 25min, using ultraviolet specrophotometer in 660nm ripple
Strong point mensuration absorbance, calculates the phosphorus content in nutrient solution using phosphorus content calibration curve.By measuring original fluid and culture
The phosphorus content in supernatant afterwards, can calculate the phosphorus decomposing of bacillus megaterium (Bacillus megaterium) 1.1123
Ability.Test shows, repeats to test through three times, dissolving P capacity reaches 61.2%.
Embodiment 4：Bacillus megaterium (Bacillus megaterium) 1.1123 shake flask fermentation is tested
Seed liquor culture medium prescription：Peptone 10.0g, beef extract 3.0g, sodium chloride 5.0g, water 1000mL.
Fermentative medium formula：Dregs of beans 6%, sucrose 2%, sodium chloride 0.3%, magnesium sulfate 0.05%, potassium dihydrogen phosphate
0.15%, balance of water.
Fermentation medium liquid amount 100mL/500mL conical flask, the bacillus megaterium that picking one ring activates
(Bacillus megaterium) 1.1123 thalline are inoculated in seed liquid culture medium, 180rpm, and 37 DEG C of culture 16h obtain and plant
Sub- liquid.Seed liquor is inoculated in fermentation medium by 5% inoculum concentration, 200rpm, 37 DEG C of culture 24h.Plate count
Method detection viable count and gemma number, calculate gemma rate.Repeat to test through three times, viable count reaches 5.6 × 109Individual/mL, gemma rate
Reach 87.5%.
Claims (3)
1. application in degraded slightly solubility or insoluble phosphorus for the bacillus megaterium (Bacillus megaterium) 1.1123.
2. bacillus megaterium (Bacillus megaterium) 1.1123 is in preparing microbial bacterial agent or microbial-bacterial fertilizer
Application.
3. a kind of microbial bacterial agent or bacterial manure are it is characterised in that with bacillus megaterium (Bacillus megaterium)
1.1123 as active ingredient.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107699518A (en) * | 2017-10-17 | 2018-02-16 | 临沂国龙生态科技有限公司 | A kind of preparation method of bacillus megaterium bacterium powder fertilizer |
| CN108676744A (en) * | 2018-05-16 | 2018-10-19 | 南京农业大学 | A kind of bacillus megaterium bacterial strain ZT-P and its application |
| CN109517756A (en) * | 2018-11-27 | 2019-03-26 | 中国科学院南海海洋研究所 | One plant of ocean phosphorus decomposing bacillus megaterium and its application |
| CN110616177A (en) * | 2019-10-29 | 2019-12-27 | 山东大学 | Bacillus with high fermentation density and fermentation production method thereof |
| CN112625941A (en) * | 2020-11-26 | 2021-04-09 | 江苏省中国科学院植物研究所 | Bacillus megaterium capable of strongly solubilizing phosphorus and application thereof |
| CN112920978A (en) * | 2021-04-15 | 2021-06-08 | 河南科技学院 | Bacillus megaterium LP17 and application thereof |
| CN113980833A (en) * | 2021-09-23 | 2022-01-28 | 中化农业（临沂）研发中心有限公司 | Bacillus megaterium and application thereof in soil phosphate solubilizing |
| CN116333905A (en) * | 2022-07-13 | 2023-06-27 | 江苏省中国科学院植物研究所 | Bacillus megaterium with functions of promoting growth and producing acid and application thereof |
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| CN102775233A (en) * | 2012-08-06 | 2012-11-14 | 定襄县神宇科技示范基地有限公司 | Novel insecticidal and antibacterial biofertilizer and preparation method thereof |
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2016
- 2016-09-14 CN CN201610824506.3A patent/CN106434436A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102775233A (en) * | 2012-08-06 | 2012-11-14 | 定襄县神宇科技示范基地有限公司 | Novel insecticidal and antibacterial biofertilizer and preparation method thereof |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107699518A (en) * | 2017-10-17 | 2018-02-16 | 临沂国龙生态科技有限公司 | A kind of preparation method of bacillus megaterium bacterium powder fertilizer |
| CN108676744A (en) * | 2018-05-16 | 2018-10-19 | 南京农业大学 | A kind of bacillus megaterium bacterial strain ZT-P and its application |
| CN108676744B (en) * | 2018-05-16 | 2020-07-28 | 南京农业大学 | Bacillus megaterium strain ZT-P and application thereof |
| CN109517756A (en) * | 2018-11-27 | 2019-03-26 | 中国科学院南海海洋研究所 | One plant of ocean phosphorus decomposing bacillus megaterium and its application |
| CN110616177A (en) * | 2019-10-29 | 2019-12-27 | 山东大学 | Bacillus with high fermentation density and fermentation production method thereof |
| CN110616177B (en) * | 2019-10-29 | 2022-06-14 | 山东大学 | Bacillus with high fermentation density and fermentation production method thereof |
| CN112625941A (en) * | 2020-11-26 | 2021-04-09 | 江苏省中国科学院植物研究所 | Bacillus megaterium capable of strongly solubilizing phosphorus and application thereof |
| CN112920978A (en) * | 2021-04-15 | 2021-06-08 | 河南科技学院 | Bacillus megaterium LP17 and application thereof |
| CN113980833A (en) * | 2021-09-23 | 2022-01-28 | 中化农业（临沂）研发中心有限公司 | Bacillus megaterium and application thereof in soil phosphate solubilizing |
| CN113980833B (en) * | 2021-09-23 | 2022-07-12 | 中化农业（临沂）研发中心有限公司 | Bacillus megaterium and application thereof in soil phosphate solubilizing |
| CN116333905A (en) * | 2022-07-13 | 2023-06-27 | 江苏省中国科学院植物研究所 | Bacillus megaterium with functions of promoting growth and producing acid and application thereof |
| CN116333905B (en) * | 2022-07-13 | 2023-09-22 | 江苏省中国科学院植物研究所 | Bacillus megaterium with functions of promoting growth and producing acid and application thereof |
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Application publication date: 20170222 |