CN1323161C - Lucerne rhizobium and its fermentation culture method and uses - Google Patents
Lucerne rhizobium and its fermentation culture method and uses Download PDFInfo
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- CN1323161C CN1323161C CNB2005101029120A CN200510102912A CN1323161C CN 1323161 C CN1323161 C CN 1323161C CN B2005101029120 A CNB2005101029120 A CN B2005101029120A CN 200510102912 A CN200510102912 A CN 200510102912A CN 1323161 C CN1323161 C CN 1323161C
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Abstract
The present invention discloses rhizobium meliloti, and a fermentation culturing method and an application thereof. The fermentation culturing method of the rhizobium meliloti (17674 CGMCC No. 1422) comprises the following steps: the rhizobium meliloti (17674 CGMCC No. 1422) is inoculated into a special liquid culturing medium, and the rhizobium meliloti is cultured in an aerobic culture mode at the temperature of 28 to 30 DEG C, wherein the special liquid culture medium comprises the following ingredients: 5 to 10g of sugar, 1 to 2g of yeast extract powder, 0.25 to 0.75g of dipotassium hydrogen phosphate, 0.16 to 0.24g of calcium sulphate, 0.1 to 0.3g of magnesium sulfate, 0.1 to 0.3g of sodium chloride, 0.5 to 1.5mL of ammonium molybdate of 1%, and 2 to 6mL of boric acid of 0.5% in each 1000 ml of water, wherein the pH value is from 6.8 to 7.0. The inoculating combination of the rhizobium meliloti (17674 CGMCC No. 1422) and meliloti can be developed into a new good combination technology in the application field of the rhizobium meliloti.
Description
Technical field
The present invention relates to a lucerne rhizobium and fermentation culture method thereof and application.
Background technology
To research and existing more than the 100 year history of application of Rhizobium leguminosarum, almost the ALFALFA PRODUCTION field in worldwide is all advocated clover is carried out legume inoculation in the world.Because the inoculation root nodule bacterium can be improved seedling rate, the seedling dross rate of clover, strengthen symbiotic nitrogen fixation, thereby improve grass yield and the protein content thereof of clover.Over past ten years, China's alfalfa industry fast development, clover inoculation root nodule bacterium The Application of Technology is all attached great importance in various places.
The problem current, that rhizobium melioti inoculation technique field exists mainly is because constantly the weeding out the old and bring forth the new of alfalfa variety, and the affinity of some clover new variety and current root nodule bacterium of applying is not strong, causes inoculating growing nitrogen-fixing DeGrain afterwards.
Attach great importance to the screening of clover symbiotic nitrogen fixation fine combination abroad.Tan carried out symbiotic nitrogen fixation fine combination shaker test with 15 alfalfa varieties and 10 lucerne rhizobiums in 1986, the result shows: the symbiotic nitrogen fixation difference between combination has 30% to be due to the alfalfa variety, 26% difference is due to the difference between the alfalfa Phylloxera bacteria strain, difference more than 36% is from the interaction between alfalfa variety and the rhizobium melioti (" Medicago in China ", Chinese agriculture press, 152 pages, 1995).In the symbiotic nitrogen fixation process, root nodule bacterium different strains and clover different varieties have very strong selectivity each other, thereby the symbiotic nitrogen fixation fine combination of screening alfalfa variety and Rhizobium strains, are the effective ways that improves clover symbiotic nitrogen fixation efficient.
Summary of the invention
The purpose of this invention is to provide root nodule bacterium and fermentation culture method thereof that a strain is used for the clover inoculation.
The rhizobium strains that is used for the clover inoculation provided by the present invention is (Rhizobium meliloti) 17674, this bacterial strain has been preserved in Chinese common micro-organisms culture presevation management committee common micro-organisms center on 07 25th, 2005, deposit number is CGMCC No.1422.
Rhizobium melioti (Rhizobium meliloti) 17674 CGMCC No.1422 belong to gram negative bacillus, no gemma, and the tool end is given birth to flagellum or peritrichous, can move.The thalline size is (0.5-0.9) * (1.0-3.0) micron, and the thalli morphology of growing in varying environment is different.Under culture condition, this bacterium is little rod-short, and thalline dyeing is inhomogeneous, forms painted and the ring bodies of coloured part not, non-staining part lipid content height.The bacterium colony of growing on yeast water mannite agar substratum plane is rounded, neat in edge, and projection slightly, colourless translucent or shallow oyster white, lawn is denseer.
Second purpose of the present invention provides the fermentation culture method of a kind of rhizobium melioti (Rhizobium meliloti) 17674 CGMCCNo.1422.
The fermentation culture method of rhizobium melioti provided by the present invention (Rhizobium meliloti) 17674 CGMCC No.1422, be that rhizobium melioti (Rhizobium meliloti) 17674 CGMCC No.1422 are inoculated in its dedicated liquid substratum, at 28-30 ℃ of following aerated culture; The prescription of described dedicated liquid substratum is: contain sugared 5-10g in every 1000mL water, yeast powder 1-2g, dipotassium hydrogen phosphate 0.25-0.75g, calcium sulfate 0.16-0.24g, sal epsom 0.1-0.3g, sodium-chlor 0.1-0.3g, 1% ammonium molybdate 0.5-1.5mL and 0.5% boric acid 2-6mL, the pH value is 6.8-7.0; Described percentage concentration is mass percent concentration.
In above-mentioned cultural method, inoculative proportion is 10-20%, is preferably 10%; The air flow of culturing process is 0.4-0.8mM O
2L
-1h
-1Cultivate and to reach the cultivation terminal point in 24-72 hour.
The prescription of preferred dedicated liquid substratum is: contain sugared 10g in every 1000mL water, yeast powder 2g, dipotassium hydrogen phosphate 0.5g, calcium sulfate 0.2g, sal epsom 0.2g, sodium-chlor 0.2g, 1% ammonium molybdate 1mL and 0.5% boric acid 4mL (solid medium adds 20g agar again), the pH value is 6.8-7.0.
Culture condition is preferably: temperature is 28 ℃, and air flow is 0.5mM O
2L
-1h
-1, incubation time is 48 hours.
Another object of the present invention provides a kind of rhizobium melioti (Rhizobium meliloti) 17674 CGMCCNo.1422 microbial inoculums.
Rhizobium melioti provided by the present invention (Rhizobium meliloti) 17674 CGMCC No.1422 microbial inoculums, its activeconstituents are rhizobium melioti (Rhizobium meliloti) 17674 CGMCC No.1422.
For obtaining better result of use, also can add the peat composed of rotten mosses in the described microbial inoculum, the fineness of the peat composed of rotten mosses is generally the 80-150 order, and the bacterium liquid of described rhizobium melioti (Rhizobium meliloti) 17674 CGMCC No.1422 and the ratio of weight and number of the peat composed of rotten mosses are 2-4: 1.
The described production method that contains rhizobium melioti (Rhizobium meliloti) the 17674 CGMCC No.1422 microbial inoculums of peat composed of rotten mosses composition, it is will be through the bacterium liquid of rhizobium melioti (Rhizobium meliloti) the 17674CGMCC No.1422 of aforesaid method fermentation and dry, aseptic, that the pH value is 6.5-7.0 peat composed of rotten mosses 2-4 by ratio of weight and the number of copies: 1 mixing, under identical condition multiplication culture 12-36 hour again, obtain containing peat composed of rotten mosses composition, and viable bacteria content is greater than rhizobium melioti (Rhizobium meliloti) the 17674 CGMCC No.1422 microbial inoculums of 200,000,000/gram; The bacterium number that contains that wherein is used for preparing the bacterium liquid of microbial inoculum is generally 3,000,000,000/mL.
Cross the 80-150 mesh sieve before the described peat composed of rotten mosses mixes with bacterium liquid, be preferably 100 mesh sieves; The condition that the peat composed of rotten mosses is sterilized can be under the 0.1 MPa pressure 1 hour.
The 4th purpose of the present invention provides a kind of method that improves the clover symbiotic nitrogen fixation.
The method of raising clover symbiotic nitrogen fixation provided by the present invention, be that alfalfa seed is inoculated above-mentioned rhizobium melioti (Rhizobium meliloti) 17674 CGMCC No.1422 or be the microbial inoculum of activeconstituents with rhizobium melioti (Rhizobium meliloti) 17674 CGMCC No.1422, through sowing, cultivation, obtain the clover that symbiotic nitrogen fixation improves again.
Be applicable to that the alfalfa variety that the present invention improves the method for symbiotic nitrogen fixation can be A Ergangjin (blue, white), American Pastoral, Huaiyin clover, No. one, middle lucerne, Baoding clover, golden clover or U.S. clover king etc.
The invention provides a lucerne rhizobium (Rhizobium meliloti) 17674 CGMCC No.1422, this bacterium and be that the microbial inoculum of activeconstituents can be used for the clover inoculation with this bacterium.After the water culture experiment proof of clover inoculation combined effect was inoculated alfalfa seed with this bacterium, alfalfa plants height and plant fresh weight increased by 56.48% and 104.93% respectively than the adjoining tree that does not connect bacterium; In the soil pot experiment, plant height and the plant fresh weight of inoculating the clover of this bacterium increase by 43.98% and 57.16% respectively than the adjoining tree that does not connect bacterium.Above-mentioned evidence rhizobium melioti (Rhizobiummeliloti) 17674 CGMCC No.1422 and clover inoculation combination have significant effect of increasing production to clover, grass yield improves can reach 49%-62%, but and have advantages of high practicability and generalization, be expected to develop into new fine combination technology in the rhizobium melioti Application Areas.The present invention has broad application prospects in clover plant husbandry.
Embodiment
Method therefor is ordinary method if no special instructions among the subordinate embodiment, and all percentage concentrations are mass percent concentration, and the solvent in all substratum is water.
The prescription of rhizobium melioti (Rhizobium meliloti) 17674 CGMCC No.1422 dedicated liquid substratum is: contain sugared 10g in every 1000mL water, yeast powder 2g, dipotassium hydrogen phosphate 0.5g, calcium sulfate 0.2g, sal epsom 0.2g, sodium-chlor 0.2g, 1% ammonium molybdate 1mL and 0.5% boric acid 4mL (solid medium adds 20g agar again), the pH value is 6.8-7.0.
Preservation and the fermentation culture of embodiment 1, rhizobium melioti (Rhizobium meliloti) 17674 CGMCC No.1422
1, the separation of bacterial classification, preservation
Gather wild alfalfa Phylloxera from Weifang, Shandong, obtain alfalfa Phylloxera bacteria strain (Rhizobiummeliloti) 17674 with the plate streaking separation, this bacterial strain has been preserved in Chinese common micro-organisms culture presevation management committee common micro-organisms center on 07 25th, 2005, deposit number is CGMCC No.1422.
2, the slant culture of alfalfa Phylloxera bacteria strain (Rhizobium meliloti) 17674 CGMCC No.1422
This bacterial classification of picking carries out slant culture to it with above-mentioned special solid substratum, cultivates 48 hours down at 28 ℃.
3, the shaking table liquid culture of bacterial classification
The rhizobium strains of picking step 2 slant culture (Rhizobium meliloti) 17674 CGMCC No.1422 are inoculated in and shake in bottle special culture solution, and 2 slant strains of every 500mL inoculation were cultivated 36 hours down at 28 ℃.
4, seed tank culture
Get the bacterium liquid of 3L step 3, insert in the 100L seeding tank (containing the 60L nutrient solution) and carry out seed culture, stir culture was cultivated in 56 hours under 30 ℃, 120rpm.
5, fermentor cultivation
In 10% ratio the seed liquor of step 4 is inserted in the 1000L fermentor tank (containing the 600L nutrient solution) and carry out fermentation culture, stir culture is 48 hours under 28 ℃, 150rpm.The gained fermented liquid can be used for the peat composed of rotten mosses and is adsorbed as the alfalfa Phylloxera bacteria agent, and the bacterium number that contains of bacterium liquid is about 3,000,000,000/mL.
Embodiment 2, contain the preparation of rhizobium melioti (Rhizobium meliloti) the 17674 CGMCC No.1422 microbial inoculums of the peat composed of rotten mosses
1, the processing of the peat composed of rotten mosses
To pulverize after the peat composed of rotten mosses seasoning, cross 100 mesh sieves, is 6.8 with the calcium lime powder adjust pH then, and sterilization is 1 hour under 0.1 MPa.
2, contain the acquisition of rhizobium melioti (Rhizobium meliloti) the 17674 CGMCC No.1422 microbial inoculums of the peat composed of rotten mosses
With 3: the 1 by ratio of weight and the number of copies mixings of the peat composed of rotten mosses that among the embodiment 1 are 6.8 through the drying of bacterium liquid and step 1 acquisition of rhizobium melioti (Rhizobium meliloti) the 17674 CGMCC No.1422 of fermentation, aseptic, pH value, again 28 ℃ of following multiplication culture 24 hours, obtain rhizobium melioti (Rhizobium meliloti) 17674 CGMCCNo.1422 microbial inoculums, but the packing warehouse-in.Finished product is qualified after testing, and viable bacteria content is greater than 200,000,000/gram.Can be used for the clover inoculation.
The test of embodiment 3, clover inoculation combined effect
1, the water culture experiment of clover inoculation combined effect
The alfalfa variety of this test usefulness is A Ergangjin (white, orchid), golden queen, American Pastoral, the shady clover in Huaihe River and Baoding clover.With the different combination of rhizobium melioti (Rhizobium meliloti) 17674 CGMCC No.1422 with above-mentioned alfalfa variety formation, carry out the water culture experiment of clover inoculation combined effect with filter paper bridge test tube water culture, be contrast with the plant that does not connect bacterium simultaneously, concrete grammar is as follows:
1) filter paper is cut into strip, its width is respectively 1.3cm, and 1.6cm makes the M type, and middle recess is made the V-type aperture according to the size of seedling, and the height of M type filter paper is that the length of test tube deducts 4cm.Used test tube is of a size of 1.5cm * 18cm.
The no nitrogen nutrition liquid that 2) will prepare is respectively charged in the test tube that has the filter paper upholder, it highly is positioned at the recess of H type filter paper, and each is handled and repeats 5 test tubes, and establishes 10 of control tube, after sealing with high voltage bearing plastics film after the can, 15 pounds 121 ℃ sterilizations 1 hour are standby.
3) select uniform alfalfa seed.Because its kind skin is thicker, wash for several times with sterilized water after 2-3 minute with vitriol oil immersion earlier, till the acid with the flush away remained on surface.
4) will place 28-30 ℃ of incubator to be incubated vernalization through the disinfectant seed.Cultivate illumination condition after the vernalization: from the top irradiation, the root lucifuge can be placed in greenhouse or the lighting box, looks after the about 7000-8000/ux of intensity; Temperature condition: 20-30 ℃.
The water culture experiment result of table 1 clover inoculation combined effect
| Combination | Plant center line average cm | Plant weight in average g | Comparison is according to increasing (%) | |
| Plant height | Plant weight | |||
| 6CMCC1422-A Ergangjin (orchid) | 9.875 | 0.204 | 74.0 | 246.97 |
| GCMCC1422-A Ergangjin (in vain) | 8.2175 | 0.109 | 29.76 | 70.31 |
| GCMCC1422--gold queen | 7.72 | 0.094 | 23.52 | 42.86 |
| The GCMCC1422-American Pastoral | 8.4 | 0.100 | 27.06 | 10.38 |
| The shady clover in GCMCC1422-Huaihe River | 8.85 | 0.117 | 126.92 | 148.94 |
| GCMCC1422-Baoding clover | 13.68 | 0.166 | 57.60 | 110.13 |
Test-results is as shown in table 1, behind rhizobium melioti (Rhizobium meliloti) 17674 CGMCC No.1422 inoculation alfalfa seed, alfalfa plants height and plant fresh weight increase by 56.48% and 104.93% respectively than the adjoining tree that does not connect bacterium, show that rhizobium melioti of the present invention (Rhizobium meliloti) 17674 CGMCC No.1422 have significant effect of increasing production to clover.
2, the soil pot experiment of clover inoculation combined effect
The alfalfa variety of this test usefulness is A Ergangjin (white, orchid), golden queen, American Pastoral, the shady clover in Huaihe River and Baoding clover.With the different combination of rhizobium melioti (Rhizobium meliloti) 17674 CGMCC No.1422 with above-mentioned alfalfa variety formation, carry out the soil pot experiment of clover inoculation combined effect, used soil pH value is 8.02, unsterilised, in solarium or greenhouse, cultivate, repeating 4 times, is contrast with the plant that does not connect bacterium simultaneously.
The soil results from pot experiment test of table 2 clover inoculation combined effect
| Combination | Plant center line average cm | Plant weight in average g | Comparison is according to increasing (%) | |
| Plant height | Plant weight | |||
| GCMCC1422-A Ergangjin (orchid) | 18.4172 | 0.21497 | 19.17 | 31.80 |
| GCMCC1422-A Ergangjin (in vain) | 23.62 | 0.2695 | 79.45 | 117.34 |
| GCMCC1422--gold queen | 14.0788 | 0.1715 | 22.96 | 54.23 |
| The GCMCC1422--American Pastoral | 16.0514 | 0.20938 | 32.05 | 15.81 |
| The shady clover in GCMCC1422--Huaihe River | 16.8292 | 0.18825 | 58.50 | 62.28 |
| GCMCC1422--Baoding clover | 23.9385 | 0.26438 | 51.77 | 61.47 |
Test-results is as shown in table 2, behind rhizobium melioti (Rhizobium meliloti) 17674 CGMCC No.1422 inoculation alfalfa seed, plant height and the plant fresh weight of inoculating the clover of this bacterium increase by 43.98% and 57.16% respectively than the adjoining tree that does not connect bacterium, show that rhizobium melioti of the present invention (Rhizobium meliloti) 17674 CGMCCNo.1422 have significant effect of increasing production to clover.
The effect comparison test of embodiment 4, rhizobium melioti (Rhizobium meliloti) 17674 CGMCC No.1422 and common application rhizobium melioti inoculation alfalfa variety
Select alfalfa variety A Ergangjin (orchid), A Ergangjin (in vain), American Pastoral, Huaiyin clover, No. one, middle lucerne and Baoding clover for use, common application rhizobium melioti ACCC17512.
Test divides three groups, one group of seed to above-mentioned alfalfa variety is not originally inoculated rhizobium melioti ACCC17512, one group of seed to above-mentioned alfalfa variety is not originally inoculated rhizobium melioti (Rhizobium meliloti) 17674 CGMCCNo.1422, and other one group is contrast, does not inoculate.According to a conventional method the seed of above-mentioned three groups of clovers is sowed, cultivate, after about 60 days, grow up to plant, write down height and the weight of each plant, and calculate the mean value of each combination, common rhizobium melioti ACCC17512 and alfalfa variety inoculation result are as shown in table 3, rhizobium melioti (Rhizobium meliloti) 17674 CGMCC No.1422 and alfalfa variety inoculation result are as shown in table 4, rhizobium melioti (Rhizobium meliloti) 17674 CGMCC No.1422 and common application rhizobium melioti ACCC1751 inoculation alfalfa variety effect comparative result are as shown in table 5, the result shows behind rhizobium melioti (Rhizobiummeliloti) the 17674 CGMCC No.1422 inoculation alfalfa seed, the alfalfa plants height is compared phenomenal growth with the plant fresh weight than the plant of the common rhizobium melioti ACCC17512 of inoculation, shows that rhizobium melioti of the present invention (Rhizobium meliloti) 17674 CGMCC No.1422 have significant effect of increasing production to clover.
Common rhizobium melioti ACCC17512 of table 3 and alfalfa variety inoculation result
| Combination | Plant center line average cm | Plant weight in average g | Comparison is according to increasing (%) | |
| Plant height | Plant weight | |||
| ACCC17512-A Ergangjin (orchid) | 7.875 | 0.199 | 38.77 | 229.03 |
| ACCC17512-A Ergangjin (in vain) | 6.33 | 0.068 | -0.047 | 6.25 |
| The ACCC17512--American Pastoral | 5.6 | 0.0608 | -15.29 | -32.89 |
| The shady clover in ACCC17512--Huaihe River | 6.4 | 0.0699 | 64.1 | 48.72 |
| No. one, lucerne among the ACCC17512-- | 5.29 | 0.0673 | -26.22 | -85.34 |
| ACCC17512--Baoding clover | 6.56 | 0.082 | -24.42 | 3.797 |
Table 4 rhizobium melioti (Rhizobium meliloti) 17674 CGMCC No.1422 and alfalfa variety connect
Plant the result
| Combination | Plant center line average cm | Plant weight in average g | Comparison is according to increasing (%) | |
| Plant height | Plant weight | |||
| GCMCC1422-A Ergangjin (orchid) | 9.875 | 0.204 | 74.0 | 246.97 |
| GCMCC1422-A Ergangjin (in vain) | 8.2175 | 0.109 | 29.76 | 70.31 |
| The GCMCC1422--American Pastoral | 8.4 | 0.1 | 27.06 | 10.38 |
| The shady clover in GCMCC1422--Huaihe River | 8.85 | 0.117 | 126.92 | 148.94 |
| No. one, lucerne among the GCMCC1422-- | 12.56 | 0.163 | 75.17 | -64.49 |
| GCMCC1422--Baoding clover | 13.68 | 0.166 | 57.6 | 110.13 |
Table 5 rhizobium melioti (Rhizobium meliloti) 17674 CGMCC No.1422 and common application Root of California Burclover
Knurl bacterium ACCC17512 inoculation alfalfa variety effect comparative result
| Combination | Plant center line average cm | Plant weight in average g | Combination | Plant center line average cm | Plant weight in average g | The plant center line average increases | The plant weight in average increases |
| GCMCC1422-A Ergangjin (orchid) | 9.875 | 0.204 | ACCC17512-A Ergangjin (orchid) | 7.875 | 0.199 | 25.39％ | 2.51％ |
| GCMCC1422-A Ergangjin (in vain) | 8.2175 | 0.109 | ACCC17512-A Ergangjin (in vain) | 6.33 | 0.068 | 29.82％ | 60.29％ |
| The GCMCC1422--American Pastoral | 8.4 | 0.1 | The ACCC17512--American Pastoral | 5.6 | 0.0608 | 50％ | 64.47％ |
| The shady clover in GCMCC1422--Huaihe River | 8.85 | 0.117 | The shady clover in ACCC17512--Huaihe River | 6.4 | 0.0699 | 38.28％ | 67.38％ |
| No. one, lucerne among the GCMCC1422-- | 12.56 | 0.163 | No. one, lucerne among the ACCC17512-- | 5.29 | 0.0673 | 137.43％ | 142.20％ |
| GCMCC1422--Baoding clover | 13.68 | 0.166 | ACCC17512--Baoding clover | 6.56 | 0.082 | 108.54％ | 102.44％ |
Claims (10)
1, rhizobium melioti (Rhizobium meliloti) 17674 CGMCC No.1422.
2, the method for a kind of fermentation culture rhizobium melioti 17674 CGMCC No.1422 is that rhizobium melioti 17674CGMCC No.1422 is inoculated in its dedicated liquid substratum, at 28-30 ℃ of following aerated culture; Described dedicated liquid substratum is made up of following substances: contain sugared 5-10g in every 1000mL water, yeast powder 1-2g, dipotassium hydrogen phosphate 0.25-0.75g, calcium sulfate 0.16-0.24g, sal epsom 0.1-0.3g, sodium-chlor 0.1-0.3g, 1% ammonium molybdate 0.5-1.5mL and 0.5% boric acid 2-6mL, the pH value is 6.8-7.0.
3, fermentation culture method according to claim 2, it is characterized in that: described dedicated liquid substratum is made up of following substances: contain sugared 10g in every 1000mL water, yeast powder 2g, dipotassium hydrogen phosphate 0.5g, calcium sulfate 0.2g, sal epsom 0.2g, sodium-chlor 0.2g, 1% ammonium molybdate 1mL and 0.5% boric acid 4mL, the pH value is 6.8-7.0.
4, according to claim 2 or 3 described fermentation culture methods, it is characterized in that: described inoculative proportion is 10-20%; The air flow of culturing process is 0.4-0.8mM O
2L
-1h
-1, incubation time is 24-72 hour.
5, fermentation culture method according to claim 4 is characterized in that: described culture condition is: temperature is 28 ℃, and air flow is 0.5mM O
2L
-1h
-1, incubation time is 48 hours.
6, be the microbial inoculum of activeconstituents with rhizobium melioti 17674 CGMCC No.1422.
7, microbial inoculum according to claim 6 is characterized in that: contain the peat composed of rotten mosses in the described microbial inoculum, the fineness of the peat composed of rotten mosses is the 80-150 order, and the bacterium liquid of described rhizobium melioti 17674 CGMCC No.1422 and the ratio of weight and number of the peat composed of rotten mosses are 2-4: 1.
8, the production method of the described microbial inoculum of a kind of claim 7, the bacterium liquid that is the rhizobium melioti 17674 CGMCC No.1422 that will obtain through the described fermentation culture method of claim 2 is 2-4 with dry, aseptic, that pH value the is 6.5-7.0 peat composed of rotten mosses by ratio of weight and the number of copies: 1 mixing, under identical condition multiplication culture 12-36 hour again, obtain containing peat composed of rotten mosses composition, and viable bacteria content is greater than the rhizobium melioti 17674 CGMCC No.1422 microbial inoculums of 200,000,000/gram; The bacterium number that contains of described bacterium liquid is: 3,000,000,000/mL.
9, production method according to claim 8 is characterized in that: cross the 80-150 mesh sieve before the described peat composed of rotten mosses mixes with bacterium liquid; The condition that the peat composed of rotten mosses is sterilized is under the 0.1 MPa pressure, 1 hour.
10, a kind of method that improves the clover symbiotic nitrogen fixation, be that inoculating the described rhizobium melioti 17674 CGMCC No.1422 of claim 1 or claim 6 alfalfa seed described is the microbial inoculum of activeconstituents with rhizobium melioti 17674 CGMCCNo.1422, through sowing, cultivation, obtain the clover that symbiotic nitrogen fixation improves again; Described alfalfa variety is A Ergangjin, American Pastoral, Huaiyin clover, No. one, middle lucerne, Baoding clover, golden clover or U.S. clover king.
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| CN105462873A (en) * | 2015-11-17 | 2016-04-06 | 北京克劳沃草业技术开发中心 | Method for making alfalfa rhizobium inoculant and rhizobium inoculant made through same and application thereof |
| CN108782094A (en) * | 2018-06-27 | 2018-11-13 | 新疆农业大学 | Rhizobium Inoculation promotes the application process of underground drip irrigation clover increasing yield and improving quality |
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| CN1156179A (en) * | 1996-12-18 | 1997-08-06 | 张龙男 | High-efficiency root-nodule bacteria for increasing yield of leguminous plant |
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| 根瘤菌属生物学特性的研究 丁彦怀 等，辽宁大学学报(自然科学版)，第21卷第1期 1994 * |
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