CN1129665C - Ammonium-resistant engigeering bacterium and biologic fertilizer containing it - Google Patents

Abstract

The present invention relates to an ammonium-resistant engineered bacterium-Enterobacter gergoviae E7 of which the preservation number is CGMCC No. 0511. The present invention also relates to a biofertilizer which comprises the ammonium-resistant engineered bacterium of the claim of right 1.

Description

A kind of engineering bacteria of anti-ammonium the and the bio-feritlizer that contains this engineering bacteria

The present invention relates to a kind of engineering bacteria of anti-ammonium the and the bio-feritlizer that contains this engineering bacteria.

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 world's model, the research heat of enclosing.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, and 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 Klebsila 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.

External laboratory makes up ooze still left alone without help the staying the greenhouse pot culture stage of research (Colnaghi et al 1997) of ammonium engineering bacteria of dissimilar anti-ammoniums, (Fujii et al 1987), (Christiansen et al1991) with genetic manipulation.1996 the rhizobium melioti with the nifA gene recombination of external source dctABD gene and modification (Rhizobium meliloti) inoculation experiments entered the land for growing field crops application stage in the U.S..Domesticly paddy rice and corn association nitrogen fixation engineering bacteria research topic have been set up from high-tech research evolutionary operation(EVOP) in 1988 863, two seminars have successfully made up the engineering bacteria of several strain different performances and have carried out the experiment of sub-district, field, during the State's Eighth Five-Year Plan period Guangdong Institute of Micro-biology has carried out hundreds thousand of mu big area inoculation tests in Jiangsu Province with paddy rice association nitrogen fixation engineering bacteria in atomic energy uses institute of Guangdong Province the Chinese Academy of Agricultural Sciences, and stimulation ratio reaches 8.4% and 5-7% respectively.

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 provide a kind of engineering bacteria of transforming through biotechnology of anti-the ammonium.

Another object of the present invention provides a kind of bio-feritlizer.

A kind of new combination azotobacter of the inventor by screening, (preserving number is: CGMCC No.0510) to collude dimension enterobacteria (Enterobacter gergoviae) 57-7 day, by the genetic engineering modified a kind of engineering bacteria of anti-ammonium the (colluding dimension enterobacteria Enterobacter gergoviaeE7 day) that obtains, 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.0511.Preservation day is on November 28th, 2000.

The characteristic of the engineering bacteria of anti-ammonium Enterobacter gergoviae E7:

1. the structure of the engineering bacteria of anti-ammonium E7:

According to the regulatory mechanism that nif among the K.pneumoniue is expressed, to express when the moulding of nifA genome, the nifA gene product of excess has been eliminated the effect of checking of nifL product, by transforming the nifA gene, makes up releasing NH ₄ ⁺The engineering bacteria of anti-ammonium that checks makes vinelandii at 10mmol/L NH ₄ ⁺More than synthetic nitrogenase.

Recombinant plasmid pCK ₃Made up by U.S. C.Kennedy laboratory, (C.Kennedy 1985) are to be integrated with pMC73A plasmid (Buchanan-Wallaston 1981a) by pRK290 plasmid (Ditta 1980) to form.The carrier of recombinant plasmid is the plasmid of pRK290 broad host range low copy number, and plasmid size has among two single restriction endonuclease sites BglII and EcoR I and the tetracyclin resistance marker gene .pRK290 integration of BglII site from pMC73A plasmid BamH IDNA fragment for 20Kb..BamH I fragment contains the anti-kalamycin gene of K.pneumoniae nifB promotor control and complete nifA gene and nifL fragment, the control of tetracycline resistance gene promotor is constitutive expression on the pRK290 plasmid because nifL promoter deletion, nifA expression of gene are subjected to.PCK ₃Recombinant plasmid has tsiklomitsin and kalamycin resistance marker, is that the plasmid of low copy number is only helping in the presence of the plasmid pRK2013 and could transform between Gram-negative bacteria, and can duplicates voluntarily in gram negative bacterium.

Recombinant plasmid pCK ₃Under the pRK2013 that helps plasmid helps,, be transformed into and obtain to deliver sub-E.gergoviae 57-7/pCK3 among the recipient bacterium E.gergoviae 57-7 by triparental cross.Delivering son repeated screening on the flat board that contains the 300mmol/L methylamine obtains anti-methylamine and has part secreting the engineering bacteria of anti-ammonium E.gergoviae E7 of ammonium ability as further experimental strain.

2. the engineering bacteria of anti-ammonium E7 characteristic:

It is identical with the growth and decline rule and the wild-type bacteria of nitrogenase activity to deliver the growth velocity of sub-E.gergoviae 57-7/pCK3 in containing antibiotic no nitrogen KP substratum, shows as the immunoblot experiment of first antibody with anti-nitrogenase ferritin antibody and delivers son at 50mmol/L NH ₄ ⁺In still can synthesize nitrogenase, acetylene reduction experiment shows delivers son at 50mmol/L NH ₄ ⁺Middle 76% nitrogenase activity that recovers recovers 52% nitrogenase activity in 100mmol/L, and wild-type bacteria is at 10mmol/L NH ₄ ⁺Just can not synthesize nitrogenase, not have nitrogenase activity.NH ₄ ⁺Influence to E.gergoviae57-7 and E.gergoviae 57-7/pCK3 nitrogenase activity:

NH 4 +Concentration	E.gergoviae 57-7/pCK3			E.gergoviae57-7
							Mmol /L	Induction time min	Nitrogenase specific activity mmol C 2H 4/ml min	Active per-cent %	Induction time hr	Nitrogenase specific activity mmol C 2H 4/ml min	Active per-cent %
0	30’	29.7	100	3.0	1.3	100
							10	30’	27.0	88	3.0	0	0
50	30’	23.4	76	3.0	0	0
							100	30’	15.1	52	-	-	-

The recombinant plasmid pCK3 that carries goal gene nifA is having resistance association to compel can stablize in recipient bacterium under the condition to duplicate and is expressing.Because of the nifA gene is that recipient bacterium itself has, the constitutive expression of nifA gene makes recipient bacterium have anti-ammonium ability, and do not having resistance association to compel under the condition recombinant plasmid pCK3 in recipient bacterium E.gergoviae 57-7 instability, pCK3 loses voluntarily with the host cell fragmentation in the continuous fragmentation process of host cell, reaches 50% and host cell fragmentation 110 generation pCK3 plasmid only retains 0.1% when the disappearance of host cell fragmentation 50 generation pCK3 in the LB substratum.

Adopt three kinds of resistances of Ap.Km.Tc monitoring works bacterium E7 simultaneously.Obtain the characteristic spectrum of E7 through Rep-PCR genome fingerprinting map spectrum analysis.

The present invention also provides a kind of bio-feritlizer, and it contains the engineering bacteria of anti-ammonium E7 of the present invention.Employed carrier can be the peat composed of rotten mosses in this bio-feritlizer.

Embodiment

At sub-district, field, Jiamusi inoculation E7, the seedling maize root system after 10 days contains E7 bacterium amount 5 * 10 ⁵CFU/ restrains dried root, rhizosphere soil 3.5 * 10 ⁵CFU/ gram soil, the outer soil 0.2 * 10 of root ³CFU/ restrains soil.The bacterium number reaches 5.6 * 10 after 20 days ⁵CFU/ restrains dried root, 6.2 * 10 ⁵CFU/ gram rhizosphere soil, 0.2 * 10 ³The outer soil of CFU/ gram root.Arrived tasseling stage, maize root system contains E7 bacterium number and drops to 3 * 10 ²CFU/ restrains native root, and rhizosphere soil contains E7 bacterium number and drops to 0.3 * 10 ²CFU/ restrains soil, and E7 bacterium number is zero in the outer soil of root, monitors the existence less than E7 in underground water always.

The survival (Jiamusi land-reclaimable crop institute 1999 year) of corn association nitrogen fixation engineering bacteria E7 in root, soil, underground water

Minute (month. day)	6.4	6.14	7.4	7.24	8.13
							Days post inoculation	10 days	20 days	40 days	60 days	80 days	110 days
Corn growth stage	The seedling phase	The seedling phase	Jointing stage	Tasseling stage	Filling stage	Ripe
							Temperature	14-23 ℃	16-25 ℃	17-27℃	21-32℃	19-31℃
The position	Bacterium number (cfu/ restrains dry ground or restrains dried root)
							Root (in Gen Biao and the root)	5.1× 10 5	5.6× 10 5	9.3×10 3	0.3×10 3	0	0
Rhizosphere soil (apart from root 1-4mm)	3.5× 10 5	6.2× 10 5	1.2×10 3	0.3×10 2	0	0
							The outer soil of root (apart from root 2-10cm)	0.2× 10 3	0.2× 10 3	0	0	0	0
Underground water (apart from 10 meters ditches of sample plot)	0	0	0	-	-	-

Inoculum size＞1 * 10 ⁷Cfu/ seed soil: meadow soil

Substratum: improvement KP substratum+Ap ¹⁰⁰, Km ⁵⁰, Tc ^12.5

Growth and decline rule at the higher Beijing sample plot E7 of temperature is identical with Jiamusi.E7 breeds along with root growth under field conditions (factors), but owing to there is not microbiotic association to compel, the easy lost part E7 of pCK3 plasmid reverts back to wild-type.

Be seeded in the corn of growing under the corn seedling cultivated under the potted plant nitrogen stress condition and sub-district, the field nitrogen fertilizer application condition with E57-7 and E7, find that inoculation increases the secondary root of Zea mays root, root system length overall and dry weight also obviously increase, and inoculation engineering bacteria E7 promotes the g and D of maize root system more significantly than inoculation wild-type E57-7.The plasmid pMGFP21 that is loaded with the nifH-gfp fusion gene is imported E57-7, and the inoculation corn seedling, owing to be subjected to the restriction of factors such as combined nitrogen, oxygen and carbon source, the nifH-gfp fusion gene can not be expressed at the corn rhizosphere.And then will be loaded with the plasmid importing E57-7/pGFP21 that can form expression nifA gene, and wherein the expression of nifH-gfp no longer is subjected to the containment of factors such as combined nitrogen, oxygen, and the engineering bacteria of anti-the ammonium has been expressed nifH-gfp on the Zea mays root surface.With ¹⁵The N isotopic dilution method also confirms, behind the corn seedling inoculation E7, have 14% to come from biological nitrogen fixation in the nitrogen that seedling absorbed in 34 days, and the fixed nitrogen percentage of inoculation E57-7 is 0.

The engineering bacteria of anti-ammonium E.gergoviae E7 and corn association nitrogen fixation

Place portion reason position	Dry weight g/ strain	Dry weight increases %	Nitrogen content %	Total nitrogen mg/ strain	15N atom % is super	Fixed nitrogen %	Amount of nitrogen fixation μ g/ strain
								The contrast root, stem and leaf	2.50	-	0.501	0.012	0.594	-	-
1.80	-	1.028	0.019	0.600	-	-
							E.gergoviae 57-7 root, stem and leaf		2.59	3.6	0.523	0.014	0.596	0.0	0
1.89	5.0	1.092	0.021	0.607	0.0	0
								E.gergoviae E7 root, stem and leaf	2.49	0	0.728	0.018	0.513	13.7	2.46
2.63	33.1	1.028	0.028	0.513	14.6	4.09

¹⁵The N isotopic dilution method, every basin 1 strain of 10 basins, 37 days seedling ages are handled in every of potted plant experiment

E7 grows in minimal medium, can be to plant hormones such as exocytosis indolylacetic acid, gibberic acid, zeatin.Engineering bacteria E7 is owing to can not only secrete plant hormone, and can continue fixed nitrogen and provide nitrogen to corn, therefore can promote the g and D of maize root system more significantly than inoculation E57-7, strengthen the ability that root system absorbs moisture and nutrition, and then the g and D of promotion plant, under suitable agronomy management, just can make corn significantly volume increase and saving nitrogenous fertilizer.

2. fungicide preparation and vaccination ways

With the KP culture medium culturing engineering bacteria E7 that contains 50 μ g/ml kantlex, 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 PH 8.0

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 are done inoculation experiments in the sub-district, field.Three kinds of application processes all have production-increasing function than aseptic contrast under normal fertilizer application condition.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.The field effect of engineering bacteria:

Continuous 4 years of 1995-1998, in Jilin Province and the land-reclaimable area, Heilongjiang Province adopt suitable farmland management condition to promote corn yield increasing significantly and saved nitrogenous fertilizer than inoculation wild-type bacteria and sterile carrier at sub-district, field inoculation engineering bacteria E7, using for further land for growing field crops provides foundation.

Inoculate the influence of the engineering bacteria of anti-ammonium E7 to corn growth

With the test of farm, Bao Quan mountain range in 1997 is example, field plot trial result at sandy loam shows, inoculation engineering bacteria E7 compares with sterile carrier with the inoculation wild-type bacteria, all is significantly increased in different growing over-ground part, underground part dry weight, and this effect is apparent in view in seedling stage.

Inoculation engineering bacteria E7 is to the influence (farm, Bao Quan mountain range 1997) of corn growth

Annotate: corn variety is four early No. 11, and overlay film is grown seedlings. rate of fertilizer application NPK=20kg/666.7m ²

Inoculation engineering bacteria E7 is to the yield increasing effect of corn

Test-results shows, no matter at soil fertility higher meadow chernozemic soil or the lower Baijiang soil of soil fertility, inoculation engineering bacteria E7 all is better than wild-type bacteria and 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 engineering bacteria E7 is all big to the influence of output than inoculation sterile carrier and wild bacterium, and inoculation engineering bacteria E7 reaches 12.3% than sterile carrier volume increase average amplitude, and inoculation engineering bacteria E7 on average increases production 8.8% than the wild bacterium E57-7 of inoculation.The biometrics of most experiments point shows that difference reaches conspicuous level (P=0.05)

Inoculation engineering bacteria E7 is to the yield increasing effect of corn in Baijiang soil, sandy loam

Time	The place	Soil type	Rate of fertilizer application NPK (kg/666.7m 2)	Handle output (kg/666.7m 2)	Volume increase % fromeCK fromeW.T
						9 9 5	The Shulan county	Baijiang soil	20	CK 574.3 E57-7 612 E7 646	0 6.5 0 12.48 * 5.6
9 9 7	Agrotechnical center, neat city	Sandy loam	20	CK 503.8 E57-7 541.6 E7 621.6	0 7.5 0 23.4 * 14.8
						9 9 7	Precious Quan Ling farm	Sandy loam	20	CK 650 E57-7 676.2 E7 738	0 4.0 0 13.5 * 9.1
9 9 7	853 farms	Baijiang soil	20	CK 679.2 E57-7 - E7 766.6	0 - - 12.8 * -
						9 9 7	The dawn farm	Baijiang soil	20	CK 717.8 E57-7 720 E7 743.3	0 0.3 0 3.6 3.2
9 9 8	Agrotechnical center, neat city	Sandy loam	16.8	CK 799.80 E57-7 782.60 E7 885.10	0 -2.1 0 10.7 * 13.1
						9 9 8	853 farms	Baijiang soil	16.8	CK 968.3 E57-7 994.3 E7 1062.1	0 2.7 0 9.7 * 6.8

^*Biometrics significant difference (P=0.05)

In black earth and chernozem experimental plot, inoculation engineering bacteria E7 reaches than sterile carrier volume increase average amplitude that 14.6% biometrics shows that difference reaches conspicuous level (P=0.05) and inoculation engineering bacteria E7 on average increases production 4.7% than the wild bacterium E57-7 of inoculation.

Inoculation engineering bacteria E7 is to the yield increasing effect of corn in black earth, chernozem

Soil Jilin Province, place	Fertilising (kg/666.7m 2)	Handle output (kg/666.7m 2)	Volume increase (%) (than CK) (than WT)
				Nine black earth (nineteen ninety-five)	NPK 16-20	CK 586.6 WT 660.0 E7 676.0 E7+K 682.6	0 12.5 0 15.2 * 2.3 16.4 *
Nine black earth (1996)	NPK 20	CK 590.6 WT 648.0 E7 672 E7+K 682.6	0 9.7 0 13.8 * 3.5 15.6 *
				Farming peace chernozem (1996)	NPK 20	CK 544 WT 573.3 E7 625.3 E7+K 646.6	0 5.4 0 15.0 * 8.3 19.0 *

It is remarkable that biometrics: F mensuration is respectively handled differences, and with L.S.D method multiple comparisons, E7 and E7+K handle, effect

All be higher than WT and CK, volume variance significantly or extremely remarkable.Except that chemical fertilizer, be affixed by farm manure 2m ³/ mu.

K is silicate bacteria (effect of composite fungus agent is better than single bacterium).

In the higher meadow chernozemic soil of fertility, when the NPK rate of fertilizer application at 20kg/666.7m ²The time, inoculation engineering bacteria E7 and wild bacterium E57-7 of inoculation and sterile carrier CK compare corn yield to influence difference little, amount of increase in production has only 2.6%.But when reducing fertilizer amount to 16.8kg/666.7m ²Time inoculation engineering bacteria E7 amount of increase in production improves.

Inoculation engineering bacteria E7 is to the yield increasing effect of corn in meadow chernozemic soil

Time	The place	Soil type	Rate of fertilizer application	Handle	Output	Volume increase %
										NPK Kg/666.7m 2		kg/666.7m 2	from CK
995	Farm, Siping City	Meadow chernozemic soil	20	CK E57-7 E7	617.4 633.1 637.4	0 2.5 3.2
							996	Pear tree	Meadow chernozemic soil	20	CK E57-7 E7	612.0 585.3 644.0	0 -4.4 5.2
997	Jiamusi crop institute	Meadow chernozemic soil	20	CK E57-7 E7	615.2 620.5 628.5	0 0.8 2.1
							998	Jiamusi crop institute	Meadow chernozemic soil	16.8	CK E57-7 E7	573.0 603.1 619.3	0 5.2 8.0

The joint fertilizer efficiency of inoculation engineering bacteria E7 is answered:

1997-1998 is that the fertile test of sub-district joint is done in the experimental plot of Baijiang soil sand boundary soil in Heilongjiang Province's soil property, saves fat proved recipe case: chemical fertilizer N: P: K=2: 1: 0.3, and NPK=16.8kg or 20kg/666.7m ²(1) contrast+100% nitrogenous fertilizer; (2) wild type strain E57-7+100% nitrogenous fertilizer; (3) wild type strain E57-7+90% nitrogenous fertilizer; (4) engineering bacteria E7+100 nitrogenous fertilizer; (5) engineering bacteria E7+90% nitrogenous fertilizer; (6) engineering bacteria E7+80% nitrogenous fertilizer.

Every processing is done seed manure with diammonium phosphate and Repone K, and amount of application is identical.The urea of executing different amounts in corn growth to the typhon mouth phase are done and are topdressed, the 100% nitrogenous fertilizer normal amount of nitrogen of making a comment or criticism, and 90%, 80% nitrogenous fertilizer refers to that amount of nitrogen is 80%, 90% of a normal amount of nitrogen.

When reducing nitrogen fertilizer amount to 90% of normal amount of nitrogen, inoculation engineering bacteria E7 still makes corn yield increasing than inoculation sterile carrier under the normal amount of nitrogen (100%N), is respectively 17.9%, 10.2%, 5.6%, 1.6% and-2.6%.And when nitrogen fertilizer amount be normal amount of nitrogen 80% the time, inoculation engineering bacteria E7 shows no increases in output than the following inoculation of normal amount of nitrogen (100%N) sterile carrier or the summary underproduction.The result shows that inoculation engineering bacteria E7 saves 15%-20% nitrogenous fertilizer than the inoculation sterile carrier on the identical yield level of corn.

When reducing nitrogen fertilizer amount to 90% of normal amount of nitrogen, inoculation engineering bacteria E7 makes corn yield increasing than the following inoculation of normal amount of nitrogen (100%N) wild-type bacteria, amount of increase in production is respectively 9.6%, 5.9%, 7.9%, 1.2% and-5.2%, and when nitrogen fertilizer amount be that 80% time inoculation engineering bacteria E7 of normal amount of nitrogen shows no increases in output or the underproduction than the following inoculation of normal amount of nitrogen (100%N) wild-type bacteria.The result shows that on the identical yield level of corn, inoculation engineering bacteria E7 saves 15% nitrogenous fertilizer than the inoculation wild-type bacteria.

The joint fertilizer efficiency of sub-district inoculation engineering bacteria E7 is answered (1997-1998) between corn field

The total amount of application 20Kg/666.7m of NPK ², soil property is a Baijiang soil, or sandy loam. ^*Biometrics significant difference (P=0.05)

Carried out 400 mu of lands for growing field crops (Baijiang soil) in the Heilongjiang Province in 1999 and inoculate engineering bacteria E7 demonstration contrast experiment, amount of increase in production reaches 7.3% (temperature influences amount of increase in production than hanging down because the Inoculant quality reaches then).

Corn association nitrogen fixation engineering bacteria E7 field demonstration experiment (1999)

The place	Neat city popularization center	Farm, Bao Quan mountain range	853 farms
				Corn variety	Four morning 11	Black 301	Pacify 307
The demonstration area	200 mu	112.5 mu	150 mu
				Soil type	Sandy loam	Sandy loam	Baijiang soil
Nitrogen fertilizer application level (kg/666.7m 2)	(12.5 urea)	(13.31 urea)	16 (urea)
				Microbial inoculum amount of application (kg/666.7m 2)	1	1	1
Handle	CK E7	CK E7	CK E7
				Mean yield (kg/666.7m 2)	374. 1402.4	586.6 620.0	465.3 504.7
Volume increase (%)	0 7.5	0 5.68	0 8.46

Microbial inoculum: E＜1 * 10 ⁹CFU/ restrains the peat composed of rotten mosses, microbial inoculum seed dressing, the live .N of corn: P: K=2: 1: 0.3

4-7 month Heilungkiang region temperature was on the low side in 1999, than low 5 ℃ of former years

Adopt the ditch inoculation of excuting a law, the E7 inoculum size is 2Kg/ microbial inoculum/666.7m, E7 bacterium number＜10 ⁹CFU/ restrains microbial inoculum

Claims (3)

1. the engineering bacteria of anti-the ammonium is characterized in that, the described engineering bacteria of anti-amine is to collude dimension enterobacteria (Enterobacter gergoviae) E7 day, and preserving number is: CGMCC No.0511.

2. a bio-feritlizer contains the described engineering bacteria of anti-ammonium of claim 1.

3. according to the described bio-feritlizer of claim 2, also contain the peat composed of rotten mosses.