CN1132930C - Azotobacterium and biologic fertilizer containing it - Google Patents
Azotobacterium and biologic fertilizer containing it Download PDFInfo
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- CN1132930C CN1132930C CN 00133626 CN00133626A CN1132930C CN 1132930 C CN1132930 C CN 1132930C CN 00133626 CN00133626 CN 00133626 CN 00133626 A CN00133626 A CN 00133626A CN 1132930 C CN1132930 C CN 1132930C
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- gergoviae
- inoculation
- bacterium
- soil
- vinelandii
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Abstract
The present invention provides an association nitrogen fixation bacterium and en-terobacter gergoviae 57-7, and a preservation number is CGMCC no. 0510. The present invention also provides a biologic fertilizer which comprises the association nitrogen fixation bacterium of the present invention. A carrier used in the biologic fertilizer of the present invention is grass peat.
Description
The present invention relates to a kind of vinelandii and the bio-feritlizer that contains these vinelandii.
From Brazilian Dobereiner laboratory in 1976 report from the grass rhizosphere separate azospirillum (Azospirillum spp), and measure azospirillum host's nitrogen supply is reached 2KgN one ha-1 d-1 (afterwards studies show that because the problem of measuring method, this numerical value is higher).Evoke worldwide research heat.Each study group separates the bacterium that multiple vinelandii (general designation combination azotobacter) comprise enterobacteriaceae, Azotobacteraceae, Bacillaceae, pseudomonas section, Spirillaceae from the gramineous crop rhizosphere in succession.But study maximum still azospirillums, the physiology of a large amount of reported in literature azospirillums, biochemistry and hereditary property have also been done a large amount of inoculation experiments with azospirillum in the field.Most importantly root nodule bacterium in the nitrogen-fixing microorganism preparation commerical prod in the world at present.With combination azotobacter Azospirillum is the Azogreen TM that the primary commercial product has only France, the Zea-Net TM of Italy, France, Belgian combination producing, these product commercial sizes of the Mexican Biofertilizonepara Maiz. of the Azo-Green of the U.S. are all little.1993 hold agricultural application with azospirillum in Uruguay is that the field inoculation experiments with azospirillum has been summed up over 20 years by the international symposium of topic, and the data summed up of symposial can reflect the overview of combination azotobacter in the field inoculation thus.The experiment in 20 years show under different areas and the climatic condition inoculation combination azotobacter to agricultural on some important crops be that production-increasing function is arranged as corn, paddy rice, wheat, though be difficult to accurately estimate the percentage ratio of inoculation experiments success, calculate with significant stimulation ratio 5-30% on the statistics, the success ratio of experiment is not 60-70% (test of some failure is reported).But the used bacterial strain of inoculation experiments all is a wild type strain so far.In microbial inoculum product a multitude of names of middle national expenditures wild-type combination azotobacter development, the good and the bad is mixed, according to statistics not following 300 families of the producer of production bio-bacterial manure.Using bacterial strain mainly is the combination azotobacter Klebsiella pneumoniae of enterobacteriaceae, bacillus and root nodule bacterium.Turnout at most and form the wheat rhizosphere association nitrogen fixation microbial inoculum that has Baoding Institute of Micro-biology to produce of product, the agent of corn rhizosphere combination azotobacter, popularizing area reaches millions of mus, but since the effect of increasing production instability stop production.
The field is inoculated and influenced by several factors, it depends on the characteristic of initial bacterium, plant variety, soil type, inoculum size and envrionment conditions, and to better bring into play the effect of microbe inoculation, one of them important step is the prescription of research Inoculant, (comprising bacterium and carrier) providing a suitable microenvironment with after guaranteeing to store at product, transport, being administered to soil, the survival rate of microbe inoculation.The most right and wrong bacilluss of combination azotobacter survival rate in soil is low also higher to keeping the conditional request of bacterial classification survival rate in the microbial inoculum.On nitragin preparation, succeeded before 10 years as the Inoculant carrier with the peat composed of rotten mosses, and extensively be applied.Because the peat composed of rotten mosses is a uncertain complicated organism, make manufacturing, store, application process has very big variability, had in nearly 10 years much and be used as the microorganism coating agent about new synthetic polymer and natural polymer, bacterial cell with these polymkeric substance parcels is made the new microbial preparation, improved the shortcoming of traditional peat composed of rotten mosses microbial inoculum, improved the survival rate in soil of microbial inoculant greatly, drought-resistant ability and biological benefit, be very potential, as the Azogreen TM of France but still there is the price height in this series products, manufacture difficulty is big, the problem high to the industrialization equipment requirements.Simultaneously a lot of researchs also show that combined inoculation agent (multiple microorganism combination) is better than single culture to the effect of plant, and this also is a kind of trend that microbiobacterial agent is produced.
An object of the present invention is to screen a kind of new vinelandii with better nitrogen fixation effect.
Another object of the present invention provides a kind of bio-feritlizer that contains new vinelandii of the present invention.
The inventor screens a kind of new combination azotobacter, collude dimension enterobacteria (Enterobactergergoviae) 57-7 day, and carried out preservation (preservation centre address: China at China Committee for Culture Collection of Microorganisms common micro-organisms center, Beijing, the Zhong Guan-cun, postcode: 100080), preserving number is: CGMCC No.0510.Preservation day is on November 28th, 2000.
Enterobacter gergoviae 57-7 characteristic:
Enterobacter gergoviae 57-7 belongs to enterobacteriaceae enterobacter (Enterrobacteriaceae) Gram-negative facultative Bacteroides nodosus, E.gergoviae 57-7 is the bacterial strain of nitrogenase activity the highest (measuring with the acetylene reduction method) from the bacterial strain that 183 strains that Guanxian county, Sichuan Zea mays root is separated to can be grown nitrogen-free agar, and classification position is to be identified and name by classification chamber Mr. Zhou Huiling of Institute of Micro-biology of the Chinese Academy of Sciences.
E.gergoviae 57-7 is Gram-negative fermented type vinelandii.Cell is rod-short, peritrichous, and motion utilizes glucose, the lactic acid aerogenesis.Do not produce acid from mucic acid, urase (+), ornithine decarboxylase (+), lysine decarboxylase (+), phenylalanine decarboxylase (-), deamination acid decarboxylase (-), indoles (-) arginine dihydrolase (-), deoxyribonuclease (-), methyl red (-), VP tests (+), 10 ℃ of growths, utilizes the Citrate trianion growth.Obtain the characteristic spectrum of E57-7 through Rep-PCR genome fingerprinting map spectrum analysis.
E.gergoviae 57-7 is well-grown on the inorganic salt nitrogen-free agar, and growth PH scope is 30 ℃ of 5-8.0 growth optimum temperutures, and growth is subjected to obvious inhibition more than 35 ℃.The E.gergoviae57-7 nitrogenase activity is being cultivated after 15 hours and is being peaked in the KP of improvement cultivates.Activity can reach 8354n mol C
2H
4/ mg albumen hour is with the souring of thalli growth substratum, NH
4 +It is synthetic to suppress nitrogenase, is not subjected to NH but nitrogenase forms the back nitrogenase activity
4 +Suppress, show the posttranslational modification mechanism that does not have nitrogenase among the E.gergoviae 57-7.
E.gergoviae 57-7 contains the big plasmid of a part amount 300Kb.And nif bunch is positioned karyomit(e).DNA hybridization and nifH:lacz determination of activity show that E.gergoviae57-7 contains nifHDK similar to pattern bacterium K.pneunoniae and nifA gene, ability and the NH of E.gergoviae 57-7 nifA gene activation nifH:lacz
4 +Relevant with oxygen.
The influence that the product of similar nifA gene is expressed the nifH:lacZ fusant among the E.gergoviae 57-7
| Bacterial strain | The host gene type | The plasmid characteristic | Betagalactosidase activity unit-O 2 +O 2(80r.p.m +NH 4 + -NH 4 + +NH 4 + -NH 4 + |
| E.gergoviae 57-7 E.gergoviae 57-7 (pSF2) K.pneumoniae Msal K.pneumoniae Msal (pSF2) | Wild-type wild-type wild-type wild-type | ----- K.p nifH：lacZ Tc rAmp r----- K.p nifH：lacZ Tc rAmp r | 24 13 5 14 61 6586 22 7992 17 16 16 15 34 2588 24 652 |
E.gergoviae57-7 can secrete plant hormone Enterobacter gergoviae 57-7 and E7 secretion plant hormone (kp liquid nutrient medium) bacterial strain plant hormone secretory volume (μ g/ml.OD660=1.0) Enterobacter gergoviae indolylacetic acid IAA Plant hormones regulators,gibberellins GA such as IAA, GA
3Zeatin Z wild-type 57-7 0.027 0.023 0.057 engineering bacteria E7 0.023 0.023 0.044
With scanning electron microscopic observation and with marker gene gfp mark E.gergoviae57-7, under fluorescent microscope, observe E.gergoviae57-7 deciding to grow and show at corn root, E.gergoviae57-7 mainly grows in the root-hair zone and secondary root crack place surely, and the paraffin section of microscopic examination immunogold silver staining is found that E.gergoviae57-7 can invade the inherent root tegumental cell gap of Zea mays root even xylem vessel is decided at the higher level but not officially announced and grown.E.gergoviae57-7 relies on numerous the prolonging of roots of plants excretory nutrition, expands with the growth of root system, also can grow the relic existence that relies on plant in rhizosphere soil surely.Can in soil, move and propagate by flowing water or dust.Dead easily at 55 ℃ of high temperature or psychrotropic bacteria below 0 ℃.E.gergoviae also can find in animal excrement, sewage, soil, is that normal enteron aisle place occupies bacterium.Institute of Epidemiology and Microbiology's check E.gergoviae 57-7 is a non-virulent pathogenic agent (table 4) through China prevention academy of sciences.The different ecological environment all has distribution to the nitrogen-fixing bacteria of enterobacteriaceae enterobacter all over the world, and have and much utilize the report of these class vinelandii, but make the report of research material only for this laboratory and Union Medical College clinical labororatory of Chinese Medical Sciences University with colluding the dimension enterobacteria day for research material.Nitrogen fixing capacity and the application of E.gergoviae reported in this laboratory first.Inst. of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine (carries out pathogenic detection to E.gergoviae57-7, and detect conclusion: this detection bacterial strain is the non-virulent pathogenic agent.Chinese Academy of Sciences heredity institute experimentation on animals center exempts to prove that from muscle and intravenous injection Da Bai E.gergoviae 57-7 does not have causes death and pathogenic with E.gergoviae 57-7 viable bacteria and dead bacterium.
The present invention also provides a kind of bio-feritlizer, and it contains of the present invention day and colludes the dimension enterobacteria.The employed carrier of this bio-feritlizer can be the peat composed of rotten mosses.
Embodiment
Fungicide preparation and vaccination ways
With KP culture medium culturing E57-7, the solid plate substratum contains 1.5% agar, and a seeding tank and a big jar substratum contain 50mmoles NH
4 +Cultivate 16-18 hour bacterium number for 30 ℃ and reach 8 * 10
9CFU/ml makes carrier with the peat composed of rotten mosses and mixes (7: 3) preparation microbial inoculum with bacterium liquid, makes the bacterium number reach 1-2 * 10
9CFU/ restrains microbial inoculum.
The KP culture medium prescription:
Na
2HPO
4.12H
2O 32g/L
KH
2PO
4 0.68g/L PH8.0
Ironic citrate 36mg/L
MgSO
4 30mg/L
CaCl
2.2H
2O 26mg/L
MnSO
4 0.3mg/L
Na
2MoO
4.2H
2O 7.6mg/L
Sucrose 20g/L
Yeast powder 0.5g/L
Base fertilizer is made in peat composed of rotten mosses microbial inoculum employing seed dressing or seed dressing adds base fertilizer, and three kinds of application processes, three kinds of application processes under normal fertilizer application condition all have production-increasing function than aseptic contrast.Calculating seed dressing by amount of increase in production adds and does base fertilizer＞do base fertilizer＞seed dressing.Amount of application is 1kg/666.7m
2, 5 * 10
7The CFU/ seed.
Inoculation E.gergoviae57-7 is to the influence of corn
With the test of farm, Bao Quan mountain range in 1997 is example, sub-district, field inoculation test result at sandy loam shows, corn inoculation wild-type bacteria is compared with sterile carrier, in different growing over-ground part, underground part dry weight increase is arranged all, and this effect is apparent in view in seedling stage.
At the lower Baijiang soil of soil fertility, inoculation E57-7 all is better than sterile carrier to the influence of corn yield in sandy loam and black earth, the chernozem, but the amplitude and the soil property of volume increase have obvious dependency.In the lower Baijiang soil of soil fertility, sandy loam, be 16.8kg/666.7m no matter use the chemical fertilizer amount
2Or 20kg/666.7m
2, inoculation E57-7 reaches 4.2% than sterile carrier volume increase average amplitude, and in black earth and chernozem experimental plot, inoculation E57-7 reaches 9.2% than sterile carrier volume increase average amplitude.
Claims (3)
1. a combination azotobacter is characterized in that, described combination azotobacter is to collude dimension enterobacteria (Enterobacter gergoviae) 57-7 day, and its preserving number is: CGMCC No.0510.
2. bio-feritlizer, it contains the described vinelandii of claim 1.
3. according to the described bio-feritlizer of claim 2, it also contains the peat composed of rotten mosses.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00133626 CN1132930C (en) | 2000-11-29 | 2000-11-29 | Azotobacterium and biologic fertilizer containing it |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00133626 CN1132930C (en) | 2000-11-29 | 2000-11-29 | Azotobacterium and biologic fertilizer containing it |
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| Publication Number | Publication Date |
|---|---|
| CN1355293A CN1355293A (en) | 2002-06-26 |
| CN1132930C true CN1132930C (en) | 2003-12-31 |
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| CN 00133626 Expired - Fee Related CN1132930C (en) | 2000-11-29 | 2000-11-29 | Azotobacterium and biologic fertilizer containing it |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101786912B (en) * | 2009-01-22 | 2012-07-18 | 湖北中烟工业有限责任公司 | High-efficient bacterial fertilizer used for tobacco floating seedlings and production method thereof |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101497541B (en) * | 2008-01-30 | 2011-11-02 | 武汉烟草(集团)有限公司 | Efficient disease-resistant phosphate solubilizing bacterial manure for tobacco and production method thereof |
| CN103509738B (en) * | 2013-07-15 | 2015-07-01 | 四川大学 | Enterobacter gergoviae and application thereof to fermentation detoxification of barbadosnut cake |
| CN108602729B (en) * | 2015-07-13 | 2022-07-05 | 皮沃特生物公司 | Methods and compositions for improving plant traits |
-
2000
- 2000-11-29 CN CN 00133626 patent/CN1132930C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101786912B (en) * | 2009-01-22 | 2012-07-18 | 湖北中烟工业有限责任公司 | High-efficient bacterial fertilizer used for tobacco floating seedlings and production method thereof |
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| Publication number | Publication date |
|---|---|
| CN1355293A (en) | 2002-06-26 |
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