CN103773709A - Bacillus subtilis with effect of efficiently dissolving phosphorus and application of bacillus subtilis - Google Patents
Bacillus subtilis with effect of efficiently dissolving phosphorus and application of bacillus subtilis Download PDFInfo
- Publication number
- CN103773709A CN103773709A CN201310474439.3A CN201310474439A CN103773709A CN 103773709 A CN103773709 A CN 103773709A CN 201310474439 A CN201310474439 A CN 201310474439A CN 103773709 A CN103773709 A CN 103773709A
- Authority
- CN
- China
- Prior art keywords
- bacillus subtilis
- phosphorus
- subtilis
- effect
- test
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The invention relates to a bacillus subtilis with the effect of efficiently dissolving phosphorus. The bacillus subtilis is preserved in the China General Microbiological Culture Collection Center and has the preservation NO. of CGMCCNo. 8241. The bacillus subtilis has the relatively high effect of dissolving the phosphorus, cannot pollute environment, and is ecological and safe. The efficient bacillus subtilis can be used independently, or used together with other organic and inorganic nutritional ingredients required by plants to be prepared into biological compound fertilizer, and which has the effects of increasing yield and improving quality.
Description
Technical field
The present invention relates to microorganism and biological fertilizer field, particularly relate to subtilis, particularly, the present invention relates to a kind of subtilis and application thereof of efficient phosphate-solubilizing effect.
Background technology
Phosphate fertilizer can promote plant bud differentiation, and early flowering result promotes seedlings root growth and improves fruit quality.Execute phosphorus and can promote various Metabolism of Normals to carry out, growth and development of plants is good, improves winter resistance and the drought resistance of plant simultaneously.Because phosphorus and metabolism and three's phase co-conversion of carbohydrate, protein and fat have relation, no matter all need phosphate fertilizer mainly with cultivation food crop, legume crop and oil crop.While lacking phosphorus, protein synthesis is obstructed, and new tenuigenin and nucleus form less, affect cell fission, and poor growth is also few, branch or the minimizing of tillering, and plant is short and small, and blade is dark green, may be that Growth of Cells is slow, and chlorophyll content improves relatively.Certain plants leaf takes on a red color or purple sometimes.Hinder sugar transportation because lack phosphorus, also oneself tired out a large amount of sugars, be conducive to the formation of yellow pigment glycosides.While lacking phosphorus, flowering period and ripening stage all postpone, yield reducation, and resistance weakens.But there is 74% the scarce phosphorus of arable soil in China, phosphorus in soil more than 95% is invalid form, plant is difficult to directly absorb, and in addition, uses in a large number chemical fertilizer obviously to cause the adverse consequencess such as soil compaction, the decline of soil water-retaining power, grassland degeneration, desertification be serious.Therefore, the utilization ratio of raising phosphorus is the focus that agronomy, soil science, ecotope and microbiological research person pay close attention to always.
People start to notice the relation between microorganism and soil phosphorus in 20 beginnings of the century.During the mixture of Sackett(1908) finding some insolublies is manured into soil, can be used as phosphorus source and applies, they filter out 50 strain bacteriums from soil, and wherein 36 strains have formed macroscopic molten phosphorus circle on flat board.Gerretsen in 1948 finds that plant applies insoluble phosphate fertilizer, after inoculation soil microorganisms, has promoted the growth of plant, increases the absorption of phosphorus.He has isolated these microorganisms, finds that these microorganisms can help the dissolving of ground phosphate rock.From then on, many scientists are devoted to the research of phosphate solubilizing bacteria, have in succession reported that many microorganisms have phosphate solubilization.
Present inventor, through concentrated research for many years, is separated to the new bacillus subtilis strain of a strain from plant rhizosphere soil, and this bacterial strain has stronger phosphate solubilization, invalid phosphorus effectively can be transformed into available phosphorus.
Summary of the invention
The object of the present invention is to provide the bacterial strain of a kind of new subtilis, described bacterial strain has good phosphate solubilization.
Subtilis of the present invention (Bacillus subtilis) has been deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 24th, 2013, depositary institution address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCC No.8241.
Further, the invention provides the purposes of above-mentioned subtilis aspect phosphorus decomposing.
Further, subtilis of the present invention can be made microbiobacterial agent, cultivates in LB nutrient solution by described bacterial strain, and culture temperature is 27 ℃, and cultivated days is 5 days, and rotating speed is 200 rpm, and nutrient solution is adjusted to 10 by bacterium liquid cell count after filtering
9individual/mL, makes subtilis microbiobacterial agent of the present invention.
Subtilis provided by the present invention separates and obtains from plant rhizosphere soil, its laboratory called after bacterial strain Fdp6.
Bacterial strain Fdp6 is rounded at the upper bacterium colony of LB solid medium (substratum consists of: Tryptones 10g/L, yeast extract 5g/L, sodium-chlor 10g/L, agar 15g/L), and edge is irregular, and oyster white is opaque, and surface irregularity has fold, tarnish.Be cultured to 30h, thalline all exists with the form of hypopus gemma.It is light yellow that bacterium colony is.Microscopic examination thalline is unicellular, shaft-like, single raw or twin, sometimes becomes chain.Gram-positive, gemma ellipse.VP reaction, Starch Hydrolysis test, nitrate reduction reaction, Citrate trianion utilization, gelatine liquefication are all positive; The utilization of the test of phenylpropionic acid desaminase, M.R., urine enzyme test, propionic salt is all negative, can be containing growing in the LB substratum of 6.5% NaCl.Utilize 16S rDNA gene specific primer to increase to 16S rDNA, amplified production is reclaimed and check order, sequencing result is carried out to Blast analysis, found that with the bacterial strain of bacterial strain Fdp6 height homology are all genus bacillus, homology is more than 98%, and in conjunction with physiological and biochemical property, identify that it is subtilis.
Effect of the present invention
In flat band method, cultivate on the solid medium that contains insoluble phosphate or organophosphorus by phosphate solubilizing bacteria, periphery of bacterial colonies produces larger molten phosphorus circle, it is 400.236 mg/L that subtilis of the present invention records its phosphorus decomposing effect through phosphorus molybdenum blue spectrophotometric method, illustrates that bacterial strain of the present invention has stronger phosphate solubilization.
Embodiment
The invention discloses a kind of efficient phosphate-solubilizing bacterium subtilis, those skilled in the art can use for reference content herein, suitably improve processing parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, these be all deemed to be included in by invention in.Subtilis of the present invention is described by the embodiment compared with standard, related personnel obviously can change methods and applications as herein described and suitable change and combination not breaking away from content of the present invention, spirit and scope, realizes and apply the technology of the present invention.
Below in conjunction with embodiment, further set forth the present invention.
the separation of embodiment 1 microorganism from soil sample
From near the Huang top chrysanthemum rhizosphere soil of adopting back the Hengshui Lake of Hebei, take 10g soil 90mL sterilized water and mix, then dilute therefrom getting 1mL sample 9mL sterilized water, carry out 6 times with this, be diluted to 10
-6, 10
-7, 10
-83 gradients, pipette bacteria suspension 100 microlitres of last three gradients, coat Meng Jinna inorganic phosphorus substratum (glucose 10 g, (NH
4)
2sO4 0.5 g, NaCl 0.3 g, KCl 0.3 g, FeSO47H2O 0.03 g, MnSO44H2O 0.03 g, MgSO47H2O 0.3 g, ground phosphate rock 10 g, yeast extract paste 0.4 g, agar 15 g, distilled water 1000 ml, pH 7.0～7.5) on flat board, with last diluent in contrast, at 28 ℃, cultivate, and every 24h observes once.Select in contrast without bacterium colony, and in diluent before this, grow the bacterial strain of bacterium colony, jump and get single bacterium colony and cultivate in liquid Meng Jinna inorganic phosphorus substratum according to the feature such as colonial morphology, color, and purifying numbering.
embodiment 2phosphorus decomposing
the screening of bacterial strain
Under gnotobasis, all inoculation, to Meng Jinna substratum, are cultivated 7 days for 30 ℃.Observe colony growth situation in flat board, filter out efficient phosphate-solubilizing bacterium according to producing phosphorus decomposing circle size.
the sequencing of the 16S rDNA of embodiment 3 bacterial strains and analysis and physiological and biochemical test are identified
Subtilis DNA extraction adopts conventional phenol method, and by bacterium universal primer amplification 16S rDNA sequence, primer sequence is 27F (AGAGTTTGATCCTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT).PCR reaction system (50 μ L) is: 10 X PCR damping fluid 5 μ L, dNTP 4 μ L, the each 1 μ L of primer, DNA profiling 2.5 μ L, Takara Taq enzyme 0.25 μ L, ultrapure water 36 μ L.Pcr amplification program is 94 ℃ of 3min; 94 ℃ of 1min, 52 ℃ of 1 min, 72 ℃ of 1.5 min, 30 circulations; 72 ℃ of 10 min.Amplified production is served Hai Sheng work bio tech ltd and is checked order.
By the 16S rDNA sequence input GenBank recording, application BLAST software carries out homology search.Choose the 16S rDNA sequence of different strains and with GenBank in known 16S rDNA carry out homology comparison.
Homology comparative result is as follows:
This pcr amplification product is carried out in GenBank to blast comparison result and show, 50 bacterial classifications of bacterial strain Fdp6 of the present invention and its height homology are all bacillus, and its homology is all more than 99%.Table 1 is depicted as 10 high bacterial strains of 16S rDNA homology of bacterial strain Fdp6.Bacterial strain physiological and biochemical index deducibility Fdp6 bacterial strain shown in associative list 2 belongs to subtilis thus.
The comparison of table 1 16S rDNA homology
| Serial ID | Bacterial strain | Homology |
| AB740156.1 | Bacillus subtilis gene for 16S rRNA, partial sequence, strain: Szi4-15 | 100% |
| KC121034.1 | Bacillus sp. JN08 16S ribosomal RNA gene, partial sequence | 100% |
| JX390617.1 | Bacillus subtilis strain NEHU.FNSRJ.113 16S ribosomal RNA gene, partial sequence | 100% |
| JQ267474.1 | Bacillus subtilis strain LY-001 16S ribosomal RNA gene, partial sequence >gb|JX849029.1| Bacillus sp. M7(2012) 16S ribosomal RNA gene, partial sequence | 100% |
| JQ023605.1 | Bacillus methylotrophicus strain LZ043 16S ribosomal RNA gene, partial sequence | 100% |
| JN400257.1 | Bacillus subtilis strain WRL-101 16S ribosomal RNA gene, partial sequence | 100% |
| EU729126.1 | Bacillus subtilis strain JSU-2 16S ribosomal RNA gene, partial sequence | 100% |
| AM981259.1 | Bacillus sp. NIOT-2 partial 16S rRNA gene, isolate NIOT-2 | 100% |
| AB848923.1 | Bacillus subtilis gene for 16S ribosomal RNA, partial sequence | 99% |
The antagonistic bacterium of screening is carried out to preliminary Physiology and biochemistry to be identified, comprise gram mensuration, catalase reaction, VP test, anaerobic growth, Starch Hydrolysis, 50 ℃ of growths and 6.5% NaCl growth test, described test method adopts the conventional methods of this class physiological and biochemical index of mensuration of this area.
The Determination of Physiological And Biochemical Indices of table 2 bacterial strain
| Testing index | Strain characteristics | Testing index | Strain characteristics |
| Gramstaining test | + | Indole test | - |
| MR test | + | VP test | - |
| Catalase test | + | H 2S produces | - |
| Nitrate reduction test | - | Sugar alcohol fermentation test | - |
| 6.5% NaCl | + | Hydrolyzed starch test | - |
| The fermentation test of glucose glycosyloxy | - |
the preparation of embodiment 4 subtilis microbiobacterial agent of the present invention
The subtilis that by deposit number is CGMCC No. 8241 is cultivated in LB nutrient solution, and culture-liquid temp is 27 ℃, and number of days is 5 days, and rotating speed is 200rpm, and pH value is 8.After fermentation culture, add 5% sucrose, then filtering and regulating bacterium liquid cell count is 10
9individual/mL, makes subtilis microbiobacterial agent of the present invention.
embodiment 5 molybdenum antimony scandium colorimetric method for determining microorganism phosphorus decomposing abilities
In phosphorous solution, add ammonium molybdate, under certain acidity condition, phosphoric acid in solution and molybdic acid complexing form yellow phosphorus molybdenum heterozygosis acid one phosphorus molybdenum Huang, then make reductive agent with xitix tartarize antimony potassium, can make phosphato-molybdic heteropolyacid change into stable blue solution (molybdenum blue), its shade and phosphorus content are proportional, can carry out thus colorimetric assay. measure the light absorption value of molybdenum blue at wavelength 700 nm places with spectrophotometer, with quantitative analysis phosphorus content.
The drafting of phosphorus typical curve
Draw successively 0.0,0.8,1.6,2.4,3.2,4.0 mL 5 mg/L phosphoric acid standardized solution and enter test tube, respectively add the anti-developer of 2 mL aluminium antimony, be settled to 20 mL with distilled water, shake up and leave standstill after 15-20 min, under 700 nm, measure absorbancy and " take absorbancy as ordinate zou; take phosphorus concentration as X-coordinate, draw phosphorus typical curve ", now in each pipe, phosphorus concentration is respectively: 0.00,0.20,0.40,0.60,0.80,1.00 mg/L.
The mensuration of titanium pigment content in nutrient solution
Get 1 mL at insoluble inorganic phosphorus liquid nutrient medium (glucose 10 g, (NH
4)
2sO
40.5 g, NaCl 0.3 g, KCl 0.3 g, MgSO
47H
2o 0.3 g, FeSO
47H
2o 0.03 g, MnSO
4h
2o 0.03 g, Ca
3(PO
4)
25.0 g, be settled to 1000 mL, pH 7.0～7.5) in cultivate thalline fermented liquid after 4d with centrifugal 10 min of 10000 g, supernatant liquor adds absorption 200 μ L and enters color board after suitably diluting, add again anti-developer 2 mL of aluminium antimony, distilled water is settled to 20 mL, leaves standstill after 15-20 min, and under 700 nm, measuring titanium pigment content is 400.236 mg/L.
A kind of efficient phosphate-solubilizing bacterium subtilis of the present invention and application thereof are described by concrete example, those skilled in the art can use for reference content of the present invention, the links such as appropriate change raw material, processing condition realize corresponding other object, its relevant change does not all depart from content of the present invention, within all similar replacements and change will become apparent to those skilled in the art that and be all deemed to be included in scope of the present invention.
Claims (3)
1. a subtilis, is characterized in that, deposit number is CGMCC No. 8241.
2. the application of the subtilis that a deposit number is CGMCC No.8241 aspect phosphorus decomposing.
3. a microbiobacterial agent, is characterized in that, subtilis claimed in claim 1 is cultivated in LB nutrient solution, and culture-liquid temp is 27 ℃, and number of days is 5 days, and rotating speed is 200 rpm, and then filtering and regulating bacterium liquid cell count is 10
9individual/mL, makes microbiobacterial agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310474439.3A CN103773709B (en) | 2013-10-12 | 2013-10-12 | Bacillus subtilis with effect of efficiently dissolving phosphorus and application of bacillus subtilis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310474439.3A CN103773709B (en) | 2013-10-12 | 2013-10-12 | Bacillus subtilis with effect of efficiently dissolving phosphorus and application of bacillus subtilis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103773709A true CN103773709A (en) | 2014-05-07 |
| CN103773709B CN103773709B (en) | 2015-05-20 |
Family
ID=50566476
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310474439.3A Expired - Fee Related CN103773709B (en) | 2013-10-12 | 2013-10-12 | Bacillus subtilis with effect of efficiently dissolving phosphorus and application of bacillus subtilis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103773709B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104195078A (en) * | 2014-08-22 | 2014-12-10 | 西安文理学院 | Bacillus subtilis |
| CN104263679A (en) * | 2014-09-03 | 2015-01-07 | 南京聚肽高科农业有限公司 | High-efficiency phosphate-solubilizing bacteria and application thereof |
| CN110591941A (en) * | 2019-08-29 | 2019-12-20 | 甘肃省科学院生物研究所 | Bacillus subtilis with efficient degradation effect on organic phosphorus and preparation method thereof |
| CN112625947A (en) * | 2020-12-16 | 2021-04-09 | 江苏省中国科学院植物研究所 | Bacillus subtilis capable of dissolving phosphorus strongly, carbon-based microbial compound fertilizer thereof and application of bacillus subtilis |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899405A (en) * | 2010-01-20 | 2010-12-01 | 辛伟成 | Bacillus subtilis GU and application in preparing efficient biological compound phosphate fertilizer thereof |
| CN102061273A (en) * | 2010-11-05 | 2011-05-18 | 南开大学 | Bacillus subtilis with phosphate-solubilizing effect and application thereof |
| CN102399713A (en) * | 2011-09-22 | 2012-04-04 | 华南农业大学 | Bacillus subtilis HL-1 and application thereof in respect of soil phosphate dissolving |
-
2013
- 2013-10-12 CN CN201310474439.3A patent/CN103773709B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899405A (en) * | 2010-01-20 | 2010-12-01 | 辛伟成 | Bacillus subtilis GU and application in preparing efficient biological compound phosphate fertilizer thereof |
| CN102061273A (en) * | 2010-11-05 | 2011-05-18 | 南开大学 | Bacillus subtilis with phosphate-solubilizing effect and application thereof |
| CN102399713A (en) * | 2011-09-22 | 2012-04-04 | 华南农业大学 | Bacillus subtilis HL-1 and application thereof in respect of soil phosphate dissolving |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104195078A (en) * | 2014-08-22 | 2014-12-10 | 西安文理学院 | Bacillus subtilis |
| CN104195078B (en) * | 2014-08-22 | 2017-01-11 | 西安文理学院 | Bacillus subtilis |
| CN104263679A (en) * | 2014-09-03 | 2015-01-07 | 南京聚肽高科农业有限公司 | High-efficiency phosphate-solubilizing bacteria and application thereof |
| CN110591941A (en) * | 2019-08-29 | 2019-12-20 | 甘肃省科学院生物研究所 | Bacillus subtilis with efficient degradation effect on organic phosphorus and preparation method thereof |
| CN112625947A (en) * | 2020-12-16 | 2021-04-09 | 江苏省中国科学院植物研究所 | Bacillus subtilis capable of dissolving phosphorus strongly, carbon-based microbial compound fertilizer thereof and application of bacillus subtilis |
| CN112625947B (en) * | 2020-12-16 | 2021-08-03 | 江苏省中国科学院植物研究所 | Bacillus subtilis capable of dissolving phosphorus strongly, carbon-based microbial compound fertilizer thereof and application of bacillus subtilis |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103773709B (en) | 2015-05-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111876351B (en) | Bacillus belgii and application thereof in relieving apple continuous cropping obstacle | |
| CN103320360B (en) | Bacillus megatherium SJ-7 strain and application thereof | |
| CN102876608B (en) | Bacillus amyloliquefaciens and application thereof | |
| CN106399177B (en) | Bacillus amyloliquefaciens and its microbial inoculum with degradation Phos and bacteriostasis | |
| CN106167776A (en) | A kind of can bacillus cereus (Bacillus cereus) TH 35 of heavy metal cadmium and application thereof in activating soil | |
| CN103627662A (en) | Peanut bradyrhizobium sp. and application thereof | |
| CN103773709B (en) | Bacillus subtilis with effect of efficiently dissolving phosphorus and application of bacillus subtilis | |
| CN114875016B (en) | Formulated carrier suitable for pseudomonas fluorescens and microbial inoculum thereof | |
| CN106222096A (en) | One strain Tabin aspergillus bacterium CT1 and the application in terms of the phosphorus decomposing of salt-soda soil thereof | |
| CN110396485B (en) | Brevibacillus brevis for producing auxin and application thereof | |
| CN109321500B (en) | Bacillus amyloliquefaciens strain and application thereof in prevention and treatment of camellia oleifera anthracnose disease | |
| CN103805518A (en) | Efficient phosphorus dissolving aspergillus niger and application thereof | |
| CN106929433B (en) | Phosphate solubilizing penicillium and application thereof | |
| CN117050913B (en) | Paenibacillus CBP-2 and application thereof | |
| CN109576171A (en) | Spindle lysine bacillus and its application | |
| CN115197853B (en) | Endophyte Epicoccum thailandicum LF-28 strain and application thereof | |
| CN106591173A (en) | Bacillus flexus HL-37 capable of activating soil heavy metal cadmium, and applications thereof | |
| CN115029249B (en) | Fungus for antagonizing potato scab and application thereof | |
| CN113817653B (en) | Pseudomonas fluorescens BsEB-1 and application thereof | |
| CN110396492A (en) | One plant of walnut branch-rot antagonistic bacterium and its application | |
| CN104805018A (en) | Agromyces sp. MT-E used for simultaneous degradation of plurality of phthalic acid esters | |
| CN105420167B (en) | A kind of Bacillus cercus and its application | |
| CN116751701B (en) | Phosphate-solubilizing bacterium and application thereof | |
| CN103060241B (en) | Acinetobacter calcoaceticus for promoting rhizosphere growth of walnuts and application thereof | |
| CN116286542B (en) | Enterobacter cloacae CBY-9 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150520 Termination date: 20181012 |