CN103805518A - Efficient phosphorus dissolving aspergillus niger and application thereof - Google Patents
Efficient phosphorus dissolving aspergillus niger and application thereof Download PDFInfo
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- CN103805518A CN103805518A CN201310474986.1A CN201310474986A CN103805518A CN 103805518 A CN103805518 A CN 103805518A CN 201310474986 A CN201310474986 A CN 201310474986A CN 103805518 A CN103805518 A CN 103805518A
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Abstract
The invention relates to efficient phosphorus dissolving aspergillus niger. The bacillus subtilis is collected with the collection number of CGMCC (China General Microbiological Culture Collection Center) No.8240 in the CGMCC. The aspergillus niger provided by the invention is relatively high in phosphorus dissolving effect, free of environment pollution and ecologically safe. A compound biofertilizer prepared by singly using the efficient aspergillus niger or using the efficient aspergillus niger and organic or inorganic nutritional ingredients required by the growth of other plants has the effects of increasing the yield and improving the quality.
Description
Technical field
The present invention relates to microorganism and biological fertilizer field, particularly relate to aspergillus niger, particularly, the present invention relates to a kind of aspergillus niger 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 Aspergillus niger 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 a kind of new Aspergillus niger strain, described bacterial strain has good phosphate solubilization.
Aspergillus niger of the present invention (Aspergillus niger) has been deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 24th, 2013, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCC No.8240.
Further, the invention provides the purposes of above-mentioned aspergillus niger aspect phosphorus decomposing.
Further, aspergillus niger of the present invention can be made microbiobacterial agent, by described bacterial strain in PD nutrient solution (200 grams of potatos, 20 grams of glucose, water 1000 mL) to cultivate, culture temperature is 27 ℃, cultivated days is 5 days, rotating speed is 200 rpm, and nutrient solution is adjusted to 10 by bacterium liquid cell count after filtering
8individual/mL, makes aspergillus niger microbiobacterial agent of the present invention.
Aspergillus niger provided by the present invention separates and obtains from plant rhizosphere soil, its laboratory called after bacterial strain Fdpa1.
Bacterial strain Fdpa1 cultivates on PDA substratum at PDA solid medium, visual inspection colony growth feature, measurement colony growth rate, optical microphotograph Microscopic observation mycelia shape and spore size, shape, type etc.Make discovery from observation, from stretch out, diameter 15～20pm, is about 1～3mm, wall thickness and smooth.Globe-roof capsule is formed on top, covers one deck metulae and one deck stigma on it comprehensively, and long on stigma have bunchiness memnonious spherical, diameter 2.5～4.0 μ m.Conidial head is spherical, diameter 700～800 μ m, brown-black.Spreading rapidly, just be white, after become aureus until black heavy fleece shape, the colourless or central slightly tawny in the back side.According to method preliminary evaluation bacterial strain Fdpa1 such as " fungi identification handbook " be aspergillus niger belong to, utilize Protocols in Molecular Biology to its ITS Sequence Identification after this bacterium be aspergillus niger Aspergillus niger.
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 537.142 mg/L that aspergillus niger 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 aspergillus niger, 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.Aspergillus niger 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 ITS sequencing of embodiment 3 bacterial strains and analysis and physiological and biochemical test are identified
ITS identifies: by the spore liquid access PD liquid nutrient medium of bacterium, 28 ℃ of shaking tables are cultivated 48 h, filter and obtain mycelia, extract fungal genomic DNA.Using extract fungal DNA as template, carry out PCR amplification with ITS5 and ITS8 primer pair.PCR reaction system is 25 μ l, and reaction system is: genomic dna 1 μ L, 10 × PCR buffer, 2.5 μ L, NS1 primer 1 μ L, NS8 primer 1 μ L, dNTPs 2 μ L, Taq enzyme 0.25 μ L, ddH2O 17.25 μ L.Reaction conditions is: 94 ℃ of 5 min, 94 ℃ of 30 s, 57 ℃ of 50 s, 72 ℃ of 50 s; Circulate 30 times; 72 ℃ of 10 min.PCR amplified production is served after Hai Sheng work Bioisystech Co., Ltd order-checking, and by the DNA sequence obtaining, input NCBI, compares analysis with all sequences in Blast program and database.
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 Fdpa1 of the present invention and its height homology are all that aspergillus niger belongs to, and its homology is all more than 99%.Show through sequential analysis, the bacterial strain that the present invention separates belongs to aspergillus niger.
the preparation of embodiment 4 aspergillus niger microbiobacterial agent of the present invention
The aspergillus niger that by deposit number is CGMCC No. 8240 is cultivated in PD 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
8individual/mL, makes aspergillus niger 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 537.142 mg/L.
A kind of efficient phosphate-solubilizing bacterium aspergillus niger 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. an aspergillus niger (Aspergillus niger), is characterized in that, deposit number is CGMCC No. 8240, and it is deposited in China Microbial Culture Preservation Commission's common micro-organisms center on September 24th, 2013.
2. the application of aspergillus niger according to claim 1 aspect soil phosphorus decomposing.
3. a microbiobacterial agent, is characterized in that, subtilis claimed in claim 1 is cultivated in PD 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
8individual/mL, makes microbiobacterial agent.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651237A (en) * | 2015-01-13 | 2015-05-27 | 青岛蔚蓝生物集团有限公司 | Efficient phosphorus-solubilizing aspergillus niger strain and application thereof |
| CN106118665A (en) * | 2016-06-08 | 2016-11-16 | 青岛蔚蓝生物集团有限公司 | A kind of compound phosphorus decomposing microbial inoculum and the application in agricultural production thereof |
| CN106754431A (en) * | 2017-02-15 | 2017-05-31 | 武汉合缘绿色生物股份有限公司 | One kind has phosphorus decomposing and the difunctional aspergillus niger of biological and ecological methods to prevent plant disease, pests, and erosion and microorganism formulation and application |
| CN113980822A (en) * | 2021-11-16 | 2022-01-28 | 青岛农业大学 | Saline-alkali-tolerant Aspergillus niger, application of Aspergillus niger as phosphate solubilizing fungus and application of Aspergillus niger in promotion of growth of saline-alkali soil corns |
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| US5912398A (en) * | 1995-06-15 | 1999-06-15 | Lockheed Idaho Technologies Company | Biomediated continuous release phosphate fertilizer |
| CN101003737A (en) * | 2007-01-17 | 2007-07-25 | 吉林农业大学 | Activator for phosphor element of soil |
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2013
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5912398A (en) * | 1995-06-15 | 1999-06-15 | Lockheed Idaho Technologies Company | Biomediated continuous release phosphate fertilizer |
| CN101003737A (en) * | 2007-01-17 | 2007-07-25 | 吉林农业大学 | Activator for phosphor element of soil |
Non-Patent Citations (1)
| Title |
|---|
| 刘晓芳 等: "一株溶磷黑曲霉的溶磷特性及溶磷机制初探", 《河南农业科学》, no. 6, 15 June 2005 (2005-06-15), pages 60 - 65 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651237A (en) * | 2015-01-13 | 2015-05-27 | 青岛蔚蓝生物集团有限公司 | Efficient phosphorus-solubilizing aspergillus niger strain and application thereof |
| CN106118665A (en) * | 2016-06-08 | 2016-11-16 | 青岛蔚蓝生物集团有限公司 | A kind of compound phosphorus decomposing microbial inoculum and the application in agricultural production thereof |
| CN106118665B (en) * | 2016-06-08 | 2019-03-26 | 青岛蔚蓝生物集团有限公司 | A kind of compound phosphorus decomposing microbial inoculum and its application in agricultural production |
| CN106754431A (en) * | 2017-02-15 | 2017-05-31 | 武汉合缘绿色生物股份有限公司 | One kind has phosphorus decomposing and the difunctional aspergillus niger of biological and ecological methods to prevent plant disease, pests, and erosion and microorganism formulation and application |
| CN113980822A (en) * | 2021-11-16 | 2022-01-28 | 青岛农业大学 | Saline-alkali-tolerant Aspergillus niger, application of Aspergillus niger as phosphate solubilizing fungus and application of Aspergillus niger in promotion of growth of saline-alkali soil corns |
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Application publication date: 20140521 |