CN103627662A - Peanut bradyrhizobium sp. and application thereof - Google Patents
Peanut bradyrhizobium sp. and application thereof Download PDFInfo
- Publication number
- CN103627662A CN103627662A CN201310643826.5A CN201310643826A CN103627662A CN 103627662 A CN103627662 A CN 103627662A CN 201310643826 A CN201310643826 A CN 201310643826A CN 103627662 A CN103627662 A CN 103627662A
- Authority
- CN
- China
- Prior art keywords
- gram
- peanut
- grams
- culture
- bradyrhizobium
- 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 bradyrhizobium sp. WD-1, as well as a culture method and application thereof. The culture method comprises slant culture, seed culture and fermentation culture, and obtains fermentation cultures containing 10 billion/ml bradyrhizobium sp. WD-1. Compared with a control peanut rhizobium, the peanut bradyrhizobium sp. WD-1 has the advantages that the peanut nodulation number is increased by 925.6%, the plant dry weight is increased by 63.6%, the plant total nitrogen content is increased by 18.1%, and the peanut yield can be increased by 57.9%. Compared with the conventional peanut rhizobium 144 used for production, the peanut Bradyrhizobium sp. WD-1 has the advantages that the nodulation number of an individual plant is increased by 27.4%, the dry weight of the individual plant is increased by 8.7%, the total nitrogen content of the individual plant is increased by 6.5%, and the yield of the individual plant is increased by 30.45%. Therefore, the bradyrhizobium sp. has broad application prospects in the peanut planting industry.
Description
[technical field]
The invention belongs to biological technical field.More specifically, the present invention relates to a kind of slow raw root nodule bacterium WD-1, also relate to the purposes of described slow raw root nodule bacterium WD-1.
[background technology]
Some prokaryotic micro-organisms of occurring in nature can synthesize nitrogenase, at normal temperatures and pressures, airborne N2 is reduced into NH3.The nitrogen of being fixed by nitrogen-fixing microorganism accounts for 65%～70% of earth's surface combined nitrogen, wherein the strongest with root nodule bacterium and leguminous plants syntaxial system nitrogen fixing capacity, accounts for the more than 65% of biological nitrogen fixation amount.Utilizing root nodule bacterium and leguminous plants symbiotic nitrogen fixation to increase soil fertility with crop yield is the classical experience of world agriculture.
Peanut is annual leguminous plants, has abundant nutritive value, and rich vitamin, protein and fat are that oil crops ， China various places of cultivated area maximum in the world all have plantation.Peanut rhizobium can form symbiosis root nodule with peanut and fix airborne nitrogen, for plant-growth provides nutrition, improve the yield and quality of peanut, can make peanut grow in poor soil, therefore, research peanut rhizobium has important practical significance and application prospect.
[summary of the invention]
[technical problem that will solve]
The object of this invention is to provide a kind of slow raw root nodule bacterium WD-1.
Another object of the present invention is to provide the purposes of described slow raw root nodule bacterium WD-1.
[technical scheme]
The present invention is achieved through the following technical solutions.
The present invention relates to a kind of slow raw root nodule bacterium (Bradyrhizobium sp.) CGMCC No.8386, this bacterial strain is on the October 23rd, 2013 of No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms center preservations in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and its preserving number is CGMCC No.8386.
The invention still further relates to the cultural method of described slow raw root nodule bacterium WD-1.
The step of this cultural method is as follows:
A, slant culture
Use transfering loop that raw root nodule bacterium WD-1 is slowly inoculated on the inclined-plane that is contained in the slant medium in test tube, be then placed in constant incubator at the condition lower inclined plane cultivation 5～7d of 28～30 ℃ of temperature, obtain slant culture;
B, seed culture
Use transfering loop to get the slant culture that a ring steps A obtains and be inoculated in liquid seed culture medium, be then placed in constant incubator, under the condition of 28～30 ℃ of temperature and rotating speed 180rpm, cultivate 4～5d, obtain inoculum;
C, fermentation culture
According to fermention medium volumeter 3%～5% inoculum size, the inoculum that step B is obtained is forwarded in liquid fermentation medium, then be placed in constant incubator and cultivate 4～5d under the condition of 28～30 ℃ of temperature and rotating speed 180rpm, the resulting fermented liquid of fermentation ends contains 10,000,000,000/ml raw root nodule bacterium WD-1 slowly.Fermented liquid is slow raw nitragin.
A preferred embodiment of the invention, the preparation method of described slant medium (solid medium) is as follows:
10～20 grams of N.F,USP MANNITOL, 0.1～1.0 gram of sodium-chlor, 0.1～1.0 gram of magnesium sulfate, 0.1～1.0 gram of calcium sulfate, 0.1～1.0 gram of dipotassium hydrogen phosphate, 1～10 gram of yeast extract paste and 18～20 grams of agar are soluble in water, toward adding 1～10ml, 1% sodium molybdate aqueous solution and 1～10ml 1% boric acid aqueous solution by weight by weight in the solution obtaining, then water supplements and reaches cumulative volume 1000ml again; Finally, use mineral acid or mineral alkali that the pH value of solution is adjusted to 6.8～7.0.
According to another kind of preferred implementation of the present invention, the preparation method of described seed culture medium is as follows:
10～20 grams of N.F,USP MANNITOL, 0.1～1 gram of sodium-chlor, 0.1～1 gram of magnesium sulfate, 0.1～1 gram of calcium sulfate, 0.1～1 gram of dipotassium hydrogen phosphate, 1～10 gram of yeast extract paste and 18～20 grams of agar are soluble in water, toward adding 1～10ml, 1% Sodium orthomolybdate and 1～10ml 1% boric acid by weight by weight in the solution obtaining, then water supplements and reaches cumulative volume 1000ml again; Finally, use mineral acid or mineral alkali that the pH value of solution is adjusted to 6.8～7.0.
According to another kind of preferred implementation of the present invention, the preparation method of described fermention medium is as follows:
1～10 gram of yeast extract paste, 5～20 grams of glycerine, 0.1～2 gram of saltpetre, 0.1～2 gram of Secondary ammonium phosphate, 0.1～1 gram of manganous sulfate, 0.1～1 gram of iron trichloride, 0.1～2 gram of dipotassium hydrogen phosphate, 0.1～2 gram of potassium primary phosphate and 0.1～1 gram of sodium-chlor is soluble in water, and then water supplements and reaches cumulative volume 1000ml; Finally, use mineral acid or mineral alkali that the pH value of solution is adjusted to 6.8～7.0.
According to another kind of preferred implementation of the present invention, described mineral acid is selected from sulfuric acid or hydrochloric acid; Described mineral alkali is selected from sodium hydroxide or potassium hydroxide.
According to another kind of preferred implementation of the present invention, the concentration of aqueous solution of described mineral acid or mineral alkali is 5-6M.
The present invention relates to the resulting slow raw nitragin of the described cultural method of a kind of employing.
The invention still further relates to described slow raw nitragin purposes in peanut cultivation.
According to another kind of preferred implementation of the present invention, in the peanut cultivation phase, described slow raw nitragin, mixes seed dressing by the consumption on 140～160ml/ mu of ground and peanut seed, then sowing.
The present invention will be described in more detail below.
The present invention relates to a kind of slow raw root nodule bacterium (Bradyrhizobium sp.) WD-1, this bacterial strain is on the October 23rd, 2013 of No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms center preservations in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and its preserving number is CGMCC No.8386.
The inventor is by isolating 20 strain wild-type peanut rhizobiums 20 soil samples from Shandong, Hebei, Henan, four peanut cultivation areas, Liaoning, after primary dcreening operation, wherein nine strains and peanut have good dross effect, through 16s order-checking, biological assay, tieback experiment, verified that these nine bacterial strains are peanut raw type root nodule bacterium slowly, through repeatedly potted plant experiment and field experiment, sieve again again, finishing screen has been selected the slow raw root nodule bacterium of a strain high-efficiency nitrogen-fixing.
Separation, the primary dcreening operation of A, peanut rhizobium (Bradyrhizobium sp.).From Shandong, Hebei, Henan, area, four, Liaoning collected the soil sample on 20 peanut cultivation ground, these 20 soil samples carried out to the seizure experiment of root nodule bacterium.
Seizure experimental technique is as follows: 10g soil-like and 90g sterilized water are added in triangular flask, and concussion shakes up, and obtains 10
-1dilution soil supension, then gets 10ml soil supension, then adds 90ml sterilized water, and concussion shakes up, and obtains 10
-2dilution soil supension, prepares 10 according to the same manner
-3, 10
-4, 10
-5dilution soil supension.These soil supensions have all carried out respectively catching experiment.
The above-mentioned soil supension of 1ml is inoculated in the peanut seed of surface sterilization in advance and rudiment, the vermiculite of usining in flowerpot is cultivated peanut seed as developing medium, regularly water conventional low nitrogen nutrient solution (vermiculite and low nitrogen nutrient solution all pass through sterilising treatment, and described low nitrogen nutrient solution is according to ordinary method preparation).After cultivating 1 month, the plant of results all has root nodule, this root nodule is carried out in solid medium to separating for several times purifying, so obtain 20 strain wild type strains.
The 20 strain wild type strains that obtain are carried out to conventional tieback experiment on peanut, to determine whether this bacterial classification and host have good dross effect, and the vermiculite using, flowerpot, pouring water, peanut seed have all carried out conventional sterilising treatment in experiment.
Through the cultivations of 2 months, observe, find that wherein 9 bacterial strains and host's peanut have good dross effect, promotion in various degree plant-growth, and other 11 bacterial strain dross effects are poor, growth-promoting effect is not obvious, some also can make crop root overstrike.Therefore, above-mentioned 9 bacterial strains have been done preservation, to carry out subsequent experimental.
B, 9 of primary dcreening operation bacterial strains have been carried out to following molecular biology identification, further determined that selected bacterial strain is peanut rhizobium.
(1) 16S rDNA sequencing:
The preparation of sample total DNA: DNA of bacteria extracting method routinely carries out.
PCR primer: forward primer 27F, reverse primer 1492R
PCR reaction system: reaction system 20 μ L volumes, reaction solution forms: 1 μ L DNA profiling, 10 μ L 2 * MastarMix, 0.4 μ L 27F, 0.4 μ L 1492R, ultrapure water complements to 20 μ L.
Pcr amplification program: 95 ℃ of 2min; 30 circulations (84 ℃ of 1min, 52 ℃ of 1min, 65 ℃ of 8min); 65 ℃ of 18min.
Amplified production send the order-checking of Beijing San Bo polygala root company after 0.7% agarose gel electrophoresis detection is qualified.After the DNA sequence dna measuring is manually corrected, use the BIAST software in NCBI to compare online, through comparison, 9 bacterial strains identifying and the similarity of rihizobium japonicum are 99%, so conclude that these 9 bacterial strains are rhizobium, in tieback experiment, there is good dross effect with peanut, so can determine that they are peanut rhizobium.
(2) Physiology and biochemistry is identified
(i), extractum carnis-peptone reaction: 9 bacterial strains are seeded in respectively in extractum carnis-protein culture medium at 28 ℃ of temperature and are cultivated 5 days.If nutrient solution clarification, thalline has no growth, identical with the contrast of inoculating strain not, and this shows that this bacterial strain can not grow on extractum carnis-protein culture medium.
Extractum carnis-protein culture medium is composed as follows: 3g extractum carnis, 10g peptone, 5g yeast extract paste, 5g sodium-chlor, 1000ml distilled water, pH7.2.
(ii), BTB reaction: 9 bacterial strains are cultivated 7 days respectively on BTB flat board at 28 ℃ of temperature.If it is blue that dull and stereotyped color becomes, illustrate that this bacterial strain produces alkali on BTB flat board, belong to slow raw type bacterial strain.
BTB reaction culture medium is composed as follows: 10g N.F,USP MANNITOL, 0.8g yeast extract powder, 0.25g dipotassium hydrogen phosphate, 0.25g potassium primary phosphate, 0.2g magnesium sulfate, 0.1g sodium-chlor, 15-20g agar, 1000ml distilled water, 1ml is 0.25% bromothymol blue by weight, pH6.8-7.0.
(iii), litmus milk reaction: 9 bacterial strains are cultivated 7 days respectively in litmus milk enrichment at 28 ℃ of temperature, and it is blue that substratum color becomes, and form whey ring, shows that this bacterial strain produces alkali, belongs to slow raw type.
Litmus milk enrichment is composed as follows: 100ml skimmed milk, 4ml2.5% reindeer moss solution, low pressure sterilizing.
(iv), 3-ketone group lactose reaction: reaction is negative, and tawny precipitation does not appear in periphery of bacterial colonies, shows that this bacterial strain is non-agrobacterium.
3-ketone group lactose medium is composed as follows: 10g lactose, 1g yeast extract powder, 1000ml distilled water, 15-20g agar, pH7.2.
By aforesaid method, identify, judge that 9 bacterial strains that screen are peanut raw type root nodule bacterium slowly.
C, potted plant screening
9 bacterial strains that primary dcreening operation obtains are by collecting region difference called after ZMW, LX, XCW, XWW, YS, YSW, ZW, DMW, WD-1, one strain Peanut Rhizobium 144 of Ling Qu our company public offering adds contrived experiment, and above-mentioned 10 bacterial strains are carried out to potted plant comparative experiments.
Experimental technique is as follows:
, according to the previously described cultural method of present specification, above-mentioned 10 bacterial strains are activated respectively and fermentation culture, cultivate the slow raw nitragin obtain standby.
(2), (for example adopt seed routine disinfection method, alcohol-pickled 5min with 95%) peanut seed surface is carried out disinfection, then sterilization peanut seed be placed on the wet gauze that soaks into sterilized water, then by its global transfer in culture dish, vernalization 7-10 days in 28 ℃ of incubators of temperature.Choose the same peanut seed of peanut sprout appearance, be soaked in respectively in the fermented liquid of 10 bacterial strains of preparation in step 1, soak 10min, then plant to being equipped with in 13 * 15cm flowerpot of vermiculite, each flowerpot is planted a strain, and each bacterial strain is designed to one group, and 8 every group parallel, separately add one group of Seed soaking in contrast, amount to 11 groups.
Draw corresponding fermented liquid and be inoculated in around seed, the OD value (λ=600nm) according to measuring bacterium liquid, guarantees during inoculation that every seedling inoculum size reaches 1.0 * 10
9more than individual.According to cultivating greenhouse illumination, temperature and humidity situation, according to peanut cultivation method, carry out Routine Management, comprising watering.
, for example, according to conventional investigation method (count knurl numbers, claim dry weight, the cooking method that disappears survey full nitrogen), when peanut plant grows into the florescence, institute an inquiry dross number, dry weight, full nitrogen, while cultivating approximately 4 months, investigate output, investigation result is if following table 1 is to table 4.
Table 1: peanut rhizobium potted plant experiment dross investigation
Table 2: peanut rhizobium potted plant experiment dry weight investigation
Table 3: the full nitrogen investigation of peanut rhizobium potted plant experiment
Table 4: peanut rhizobium experimental yield investigation
By above-mentioned table 1, to the experimental result of table 4, can be found out, peanut rhizobium (Bradyrhizobium sp.) WD-1 has obvious growth promoting function, follows other bacterial strain to compare with the obvious advantage, and is better than existing bacterial strain.
By relatively, to use the peanut plant of peanut rhizobium (Bradyrhizobium sp.) WD-1 bacterium liquid and compared with contrast, individual nodule number improves 925.6%, and total dry weight improves 63.6%, and individual plant total nitrogen content improves 18.1%, and single plant yield improves 57.9%.
Compare with the peanut rhizobium 144 of existing production and application, individual nodule number improves 27.4%, and total dry weight improves 8.7%, and individual plant total nitrogen content improves 6.5%, and single plant yield improves 30.45%.
Through biological assay, tieback experiment, verified that these nine bacterial strains are peanut raw type root nodule bacterium slowly, through repeatedly potted plant experiment and field experiment, sieve again again, finishing screen has been selected the Peanut Rhizobium of a strain high-efficiency nitrogen-fixing, i.e. raw root nodule bacterium (Bradyrhizobium sp.) WD-1 slowly.
By microscope and flat-plate bacterial colony, observe, raw root nodule bacterium WD-1 belongs to negative bacillus slowly, and the raw flagellum of peritrichous or end, can move, without gemma.Thalline size is (0.5-0.9) * (1.0-3.0) micron, at different growing environments and different growth phase, comes in every shape, and under liquid culture condition, it is tiny shaft-like that this bacterium becomes, and thalline is painted inhomogeneous.Under the dull and stereotyped condition of solid culture, while cultivating 5-7 days, the circular projection of bacterium colony, diameter 0.5-1 millimeter, edge is whole strange, and bacterium colony becomes oyster white translucent, surface wettability.
The 16S rDNA complete sequence of this slow raw root nodule bacterium WD-1 is shown in appendix 1.
The invention still further relates to the cultural method of described slow raw root nodule bacterium WD-1.
The step of this cultural method is as follows:
A, slant culture
Use transfering loop that raw root nodule bacterium WD-1 is slowly inoculated on the inclined-plane that is contained in the slant medium in test tube, be then placed in constant incubator at the condition lower inclined plane cultivation 5～7d of 28～30 ℃ of temperature, obtain slant culture.
The preparation method of described slant medium is as follows:
10～20 grams of N.F,USP MANNITOL, 0.1～1.0 gram of sodium-chlor, 0.1～1.0 gram of magnesium sulfate, 0.1～1.0 gram of calcium sulfate, 0.1～1.0 gram of dipotassium hydrogen phosphate, 1～10 gram of yeast extract paste and 18～20 grams of agar are soluble in water, toward adding 1～10ml, 1% sodium molybdate aqueous solution and 1～10ml 1% boric acid aqueous solution by weight by weight in the solution obtaining, then water supplements and reaches cumulative volume 1000ml again; Finally, use mineral acid or mineral alkali that the pH value of solution is adjusted to 6.8～7.0.
In this step and subsequent step, the constant incubator of use is all product sold in the market.
In this step and subsequent step, described mineral acid is selected from sulfuric acid or hydrochloric acid; Described mineral alkali is selected from sodium hydroxide or potassium hydroxide.
The mineral acid using is inorganic acid aqueous solution, and mineral alkali is inorganic base aqueous solution.The concentration of the described aqueous solution is 5-6M normally.
B, seed culture
Use the slant culture that transfering loop obtains a ring steps A to be inoculated in liquid seed culture medium, be then placed in constant incubator and cultivate 4～5d under the condition of 28～30 ℃ of temperature and rotating speed 180rpm, obtain inoculum.
The preparation method of described seed culture medium is as follows:
10～20 grams of N.F,USP MANNITOL, 0.1～1 gram of sodium-chlor, 0.1～1 gram of magnesium sulfate, 0.1～1 gram of calcium sulfate, 0.1～1 gram of dipotassium hydrogen phosphate, 1～10 gram of yeast extract paste and 18～20 grams of agar are soluble in water, toward adding 1～10ml, 1% Sodium orthomolybdate and 1～10ml 1% boric acid by weight by weight in the solution obtaining, then water supplements and reaches cumulative volume 1000ml again; Finally, use mineral acid or mineral alkali that the pH value of solution is adjusted to 6.8～7.0.
C, fermentation culture
According to fermention medium volumeter 3%～5% inoculum size, the inoculum that step B is obtained is forwarded in liquid fermentation medium, then be placed in constant incubator and cultivate 4～5d under the condition of 28～30 ℃ of temperature and rotating speed 180rpm, the resulting fermenting culture of fermentation ends contains 10,000,000,000/ml raw root nodule bacterium WD-1 slowly.
The preparation method of described fermention medium is as follows:
1～10 gram of yeast extract paste, 5～20 grams of glycerine, 0.1～2 gram of saltpetre, 0.1～2 gram of Secondary ammonium phosphate, 0.1～1 gram of manganous sulfate, 0.1～1 gram of iron trichloride, 0.1～2 gram of dipotassium hydrogen phosphate, 0.1～2 gram of potassium primary phosphate and 0.1～1 gram of sodium-chlor is soluble in water, and then water supplements and reaches cumulative volume 1000ml; Finally, use mineral acid or mineral alkali that the pH value of solution is adjusted to 6.8～7.0.
The method of counting of the slow raw root nodule bacterium WD-1 content of resulting fermented liquid is the method that adopts viable bacteria plate count conventional in the art.
The present invention relates to a kind of slow raw nitragin.This microbial inoculum fermented liquid that aforesaid method prepares for adopting, that contain 10,000,000,000/ml slow raw root nodule bacterium WD-1 alive.
The invention still further relates to described slow raw nitragin purposes in peanut cultivation.
Described slow raw nitragin using method is as follows:
In the peanut cultivation phase, the slow raw nitragin obtaining according to the above-mentioned knurl bacteria fermentation preparation method of taking root slowly, mixes seed dressing by the consumption on 140～160ml/ mu of ground and peanut seed, then sowing.
[beneficial effect]
The invention has the beneficial effects as follows: the invention provides a strain peanut rhizobium (Bradyrhizobium sp.) WD-1.Experimental results show that peanut plant was used the growth of the microbial inoculum that this bacterial strain makes vigorous, compared with the control, use the peanut dross number of peanut rhizobium WD-1 of the present invention to improve 925.6%, plant dry weight improves 63.6%, total nitrogen improves 18.1%, and peanut yield can improve 57.9%.Compare with the peanut rhizobium 144 of existing production and application, individual nodule number improves 27.4%, and total dry weight improves 8.7%, and individual plant total nitrogen content improves 6.5%, and single plant yield improves 30.45%.Therefore the present invention has broad application prospects in peanut cultivation industry.
Slow raw root nodule bacterium (Bradyrhizobium sp.) WD-1, this bacterial strain is on the October 23rd, 2013 of No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms center preservations in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and its preserving number is CGMCC No.8386.
[embodiment]
By following embodiment, can understand better the present invention.
The experimental technique using in embodiment below if no special instructions, is ordinary method.
The material that used in the following embodiments, reagent, instrument etc., if no special instructions, all can buy and obtain from commercial channels.
Embodiment 1: peanut rhizobium WD-1 cultivates and application
The implementation step of this embodiment is as follows:
A, slant culture
First, prepare slant medium:
10 grams of N.F,USP MANNITOL, 0.1 gram of sodium-chlor, 0.6 gram of magnesium sulfate, 0.1 gram of calcium sulfate, 0.3 gram of dipotassium hydrogen phosphate, 10 grams of yeast extract pastes and 18 grams of agar are soluble in water, toward adding 8ml 1% sodium molybdate aqueous solution and 1ml 1% boric acid aqueous solution by weight by weight in the solution obtaining, then water supplements and reaches cumulative volume 1000ml again; The aqueous solution obtaining is finally used 5M sodium hydroxide or 6M aqueous sulfuric acid that its pH value is adjusted to 6.8.
Then, use transfering loop that raw root nodule bacterium WD-1 is slowly inoculated on the inclined-plane that is contained in slant medium described in test tube, be then placed in constant incubator at the condition lower inclined plane cultivation 5d of 30 ℃ of temperature, obtain slant culture;
B, seed culture
First, prepare seed culture medium:
14 grams of N.F,USP MANNITOL, 0.3 gram of sodium-chlor, 0.1 gram of magnesium sulfate, 0.6 gram of calcium sulfate, 0.1 gram of dipotassium hydrogen phosphate, 8 grams of yeast extract pastes and 19 grams of agar are soluble in water, toward adding 4ml 1% Sodium orthomolybdate and 1ml 1% boric acid by weight by weight in the solution obtaining, then water supplements and reaches cumulative volume 1000ml again; The aqueous solution obtaining is used 5M sodium hydroxide or 6M aqueous sulfuric acid that its pH value is adjusted to 6.9.
Then, use the slant culture that transfering loop obtains a ring steps A to be inoculated in liquid seed culture medium, be then placed in constant incubator and cultivate 4d under the condition of 28 ℃ of temperature and rotating speed 180rpm, obtain inoculum;
C, fermentation culture
First, prepare fermention medium:
3 grams of yeast extract pastes, 20 grams of glycerine, 0.8 gram of saltpetre, 1.4 grams of Secondary ammonium phosphates, 0.1 gram of manganous sulfate, 0.6 gram of iron trichloride, 0.8 gram of dipotassium hydrogen phosphate, 0.8 gram of potassium primary phosphate and 0.3 gram of sodium-chlor is soluble in water, and then water supplements and reaches cumulative volume 1000ml; Finally, use 5M sodium hydroxide or 6M aqueous sulfuric acid that the pH value of solution is adjusted to 6.8.
Then, according to fermention medium volumeter 4% inoculum size, the inoculum that step B is obtained is forwarded in liquid fermentation medium, then be placed in constant incubator and cultivate 4d under the condition of 28 ℃ of temperature and rotating speed 180rpm, the resulting fermenting culture of fermentation ends is slow raw nitragin.
Adopt the method for viable bacteria plate count to identify, described slow raw nitragin contains 10,500,000,000/ml slow raw root nodule bacterium WD-1 alive.
The slow raw nitragin that uses the present embodiment to prepare, the method that adopts this specification sheets to describe has been carried out peanut plantation test, measures 396 of individual nodule numbers, 4.33 grams of total dry weights, individual plant total nitrogen content by weight 2.81%, 26/strain of individual plant peanut yield.
Embodiment 2: peanut rhizobium WD-1 cultivates and application
The implementation step of this embodiment is as follows:
A, slant culture
First, prepare slant medium:
14 grams of N.F,USP MANNITOL, 0.3 gram of sodium-chlor, 1.0 grams of magnesium sulfate, 1.0 grams of calcium sulfate, 0.6 gram of dipotassium hydrogen phosphate, 1 gram of yeast extract paste and 19 grams of agar are soluble in water, toward adding 10ml 1% sodium molybdate aqueous solution and 4ml 1% boric acid aqueous solution by weight by weight in the solution obtaining, then water supplements and reaches cumulative volume 1000ml again; Finally, use 6M sodium hydroxide or 5M aqueous sulfuric acid that the pH value of solution is adjusted to 6.9.
Then, use transfering loop that raw root nodule bacterium WD-1 is slowly inoculated on the inclined-plane that is contained in the slant medium in test tube, be then placed in constant incubator at the condition lower inclined plane cultivation 6d of 28 ℃ of temperature, obtain slant culture;
B, seed culture
First, prepare seed culture medium:
16 grams of N.F,USP MANNITOL, 0.6 gram of sodium-chlor, 1.0 grams of magnesium sulfate, 1.0 grams of calcium sulfate, 0.3 gram of dipotassium hydrogen phosphate, 10 grams of yeast extract pastes and 18 grams of agar are soluble in water, toward adding 1ml 1% Sodium orthomolybdate and 4ml 1% boric acid by weight by weight in the solution obtaining, then water supplements and reaches cumulative volume 1000ml again; Finally, use 6M potassium hydroxide or 5M aqueous sulfuric acid that the pH value of solution is adjusted to 6.8.
Then, use the slant culture that transfering loop obtains a ring steps A to be inoculated in liquid seed culture medium, be then placed in constant incubator and cultivate 5d under the condition of 28 ℃ of temperature and rotating speed 180rpm, obtain inoculum;
C, fermentation culture
First, prepare fermention medium:
8 grams of yeast extract pastes, 5 grams of glycerine, 0.1 gram of saltpetre, 0.6 gram of Secondary ammonium phosphate, 0.3 gram of manganous sulfate, 1.0 grams of iron trichlorides, 0.1 gram of dipotassium hydrogen phosphate, 0.1 gram of potassium primary phosphate and 0.6 gram of sodium-chlor is soluble in water, and then water supplements and reaches cumulative volume 1000ml; Finally, use 6M potassium hydroxide or 5M aqueous sulfuric acid that the pH value of solution is adjusted to 6.9.
Then, according to fermention medium volumeter 3% inoculum size, the inoculum that step B is obtained is forwarded in liquid fermentation medium, then be placed in constant incubator and cultivate 5d under the condition of 28 ℃ of temperature and rotating speed 180rpm, the resulting fermenting culture of fermentation ends is slow raw nitragin.
Adopt the method for viable bacteria plate count to identify, described slow raw nitragin contains 9,800,000,000/ml slow raw root nodule bacterium WD-1 alive.
The slow raw nitragin that uses the present embodiment to prepare, the method that adopts this specification sheets to describe has been carried out peanut plantation test, measure 354 of individual nodule numbers, 4.32 grams of total dry weights, individual plant total nitrogen content by weight 2.66% with 28/strain of individual plant peanut yield.
Embodiment 3: peanut rhizobium WD-1 cultivates and application
The implementation step of this embodiment is as follows:
A, slant culture
First, prepare slant medium:
16 grams of N.F,USP MANNITOL, 0.6 gram of sodium-chlor, 1.0 grams of magnesium sulfate, 1.0 grams of calcium sulfate, 0.6 gram of dipotassium hydrogen phosphate, 3 grams of yeast extract pastes and 20 grams of agar are soluble in water, toward adding 1ml 1% sodium molybdate aqueous solution and 8ml 1% boric acid aqueous solution by weight by weight in the solution obtaining, then water supplements and reaches cumulative volume 1000ml again; Finally, use 5.5M sodium hydroxide or 6M aqueous hydrochloric acid that the pH value of solution is adjusted to 7.0.
Then, use transfering loop that raw root nodule bacterium WD-1 is slowly inoculated on the inclined-plane that is contained in the slant medium in test tube, be then placed in constant incubator at the condition lower inclined plane cultivation 7d of 29 ℃ of temperature, obtain slant culture;
B, seed culture
First, prepare seed culture medium:
10 grams of N.F,USP MANNITOL, 0.1 gram of sodium-chlor, 0.3 gram of magnesium sulfate, 0.1 gram of calcium sulfate, 0.6 gram of dipotassium hydrogen phosphate, 1 gram of yeast extract paste and 20 grams of agar are soluble in water, toward adding 6ml 1% Sodium orthomolybdate and 8ml 1% boric acid by weight by weight in the solution obtaining, then water supplements and reaches cumulative volume 1000ml again; Finally, use 5.5M potassium hydroxide or 6M aqueous sulfuric acid that the pH value of solution is adjusted to 6.9.
Then, use the slant culture that transfering loop obtains a ring steps A to be inoculated in liquid seed culture medium, be then placed in constant incubator and cultivate 4d under the condition of 29 ℃ of temperature and rotating speed 180rpm, obtain inoculum;
C, fermentation culture
First, prepare fermention medium:
10 grams of yeast extract pastes, 10 grams of glycerine, 1.4 grams of saltpetre, 0.1 gram of Secondary ammonium phosphate, 0.6 gram of manganous sulfate, 0.1 gram of iron trichloride, 1.4 grams of dipotassium hydrogen phosphates, 1.4 grams of potassium primary phosphates and 0.1 gram of sodium-chlor is soluble in water, and then water supplements and reaches cumulative volume 1000ml; Finally, use 5.5M sodium hydroxide or 6M aqueous hydrochloric acid that the pH value of solution is adjusted to 7.0.
Then, according to fermention medium volumeter 4% inoculum size, the inoculum that step B is obtained is forwarded in liquid fermentation medium, then be placed in constant incubator and cultivate 4d under the condition of 29 ℃ of temperature and rotating speed 180rpm, the resulting fermenting culture of fermentation ends is slow raw nitragin.
Adopt viable bacteria plate count method to identify, described slow raw nitragin contains 102.5 hundred million/ml slow raw root nodule bacterium WD-1 alive.
The slow raw nitragin that uses the present embodiment to prepare, the method that adopts this specification sheets to describe has been carried out peanut plantation test, measures 412 of individual nodule numbers, 4.42 grams of total dry weights, individual plant total nitrogen content by weight 2.79%, 33/strain of individual plant peanut yield.
Embodiment 4: peanut rhizobium WD-1 cultivates and application
The implementation step of this embodiment is as follows:
A, slant culture
First, prepare slant medium:
20 grams of N.F,USP MANNITOL, 1.0 grams of sodium-chlor, 0.3 gram of magnesium sulfate, 0.6 gram of calcium sulfate, 1.0 grams of dipotassium hydrogen phosphates, 8 grams of yeast extract pastes and 19 grams of agar are soluble in water, toward adding 4ml 1% sodium molybdate aqueous solution and 10ml 1% boric acid aqueous solution by weight by weight in the solution obtaining, then water supplements and reaches cumulative volume 1000ml again; Finally, use 5M sodium hydroxide or 6M aqueous sulfuric acid that the pH value of solution is adjusted to 6.8.
Then, use transfering loop that raw root nodule bacterium WD-1 is slowly inoculated on the inclined-plane that is contained in the slant medium in test tube, be then placed in constant incubator at the condition lower inclined plane cultivation 6d of 29 ℃ of temperature, obtain slant culture;
B, seed culture
First, prepare seed culture medium:
20 grams of N.F,USP MANNITOL, 1.0 grams of sodium-chlor, 0.6 gram of magnesium sulfate, 0.3 gram of calcium sulfate, 1.0 grams of dipotassium hydrogen phosphates, 3 grams of yeast extract pastes and 19 grams of agar are soluble in water, toward adding 10ml 1% Sodium orthomolybdate and 10ml 1% boric acid by weight by weight in the solution obtaining, then water supplements and reaches cumulative volume 1000ml again; Finally, use 5M sodium hydroxide or 6M aqueous sulfuric acid that the pH value of solution is adjusted to 7.0.
Then, use the slant culture that transfering loop obtains a ring steps A to be inoculated in liquid seed culture medium, be then placed in constant incubator and cultivate 5d under the condition of 30 ℃ of temperature and rotating speed 180rpm, obtain inoculum;
C, fermentation culture
First, prepare fermention medium:
1 gram of yeast extract paste, 15 grams of glycerine, 2 grams of saltpetre, 2 grams of Secondary ammonium phosphates, 1.0 grams of manganous sulfates, 0.3 gram of iron trichloride, 2 grams of dipotassium hydrogen phosphates, 2 grams of potassium primary phosphates and 1.0 grams of sodium-chlor are soluble in water, and then water supplements and reaches cumulative volume 1000ml; Finally, use 5M sodium hydroxide or 5M aqueous sulfuric acid that the pH value of solution is adjusted to 6.8.
Then, according to fermention medium volumeter 5% inoculum size, the inoculum that step B is obtained is forwarded in liquid fermentation medium, then be placed in constant incubator and cultivate 5d under the condition of 30 ℃ of temperature and rotating speed 180rpm, the resulting fermenting culture of fermentation ends is slow raw nitragin.
Adopt viable bacteria plate count method to identify, described slow raw nitragin contains 10,300,000,000/ml slow raw root nodule bacterium WD-1 alive.
The slow raw nitragin that uses the present embodiment to prepare, the method that adopts this specification sheets to describe has been carried out peanut plantation test, measure 389 of individual nodule numbers, 4.22 grams of total dry weights, individual plant total nitrogen content by weight 2.57% with 29/strain of individual plant peanut yield.
Claims (10)
1. slow raw root nodule bacterium (Bradyrhizobium sp.) WD-1, this bacterial strain is on the October 23rd, 2013 of No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms center preservations in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and its preserving number is CGMCC No.8386.
2. the cultural method of slow raw root nodule bacterium WD-1 according to claim 1, is characterized in that the step of the method is as follows:
A, slant culture
Use transfering loop that raw root nodule bacterium WD-1 is slowly inoculated on the inclined-plane that is contained in the slant medium in test tube, be then placed in constant incubator at the condition lower inclined plane cultivation 5～7d of 28～30 ℃ of temperature, obtain slant culture;
B, seed culture
Use the slant culture that transfering loop obtains a ring steps A to be inoculated in liquid seed culture medium, be then placed in constant incubator, under the condition of 28～30 ℃ of temperature and rotating speed 180rpm, cultivate 4～5d, obtain inoculum;
C, fermentation culture
According to fermention medium volumeter 3%～5% inoculum size, the inoculum that step B is obtained is forwarded in liquid fermentation medium, then be placed in constant incubator and cultivate 4～5d under the condition of 28～30 ℃ of temperature and rotating speed 180rpm, the resulting fermenting culture of fermentation ends contains 10,000,000,000/ml raw root nodule bacterium WD-1 slowly.
3. cultural method according to claim 2, is characterized in that the preparation method of described slant medium is as follows:
10～20 grams of N.F,USP MANNITOL, 0.1～1.0 gram of sodium-chlor, 0.1～1.0 gram of magnesium sulfate, 0.1～1.0 gram of calcium sulfate, 0.1～1.0 gram of dipotassium hydrogen phosphate, 1～10 gram of yeast extract paste and 18～20 grams of agar are soluble in water, toward adding 1～10ml, 1% sodium molybdate aqueous solution and 1～10ml 1% boric acid aqueous solution by weight by weight in the solution obtaining, then water supplements and reaches cumulative volume 1000ml again; Finally, use mineral acid or mineral alkali that the pH value of solution is adjusted to 6.8～7.0.
4. cultural method according to claim 2, is characterized in that the preparation method of described seed culture medium is as follows:
10～20 grams of N.F,USP MANNITOL, 0.1～1 gram of sodium-chlor, 0.1～1 gram of magnesium sulfate, 0.1～1 gram of calcium sulfate, 0.1～1 gram of dipotassium hydrogen phosphate, 1～10 gram of yeast extract paste and 18～20 grams of agar are soluble in water, toward adding 1～10ml, 1% Sodium orthomolybdate and 1～10ml 1% boric acid by weight by weight in the solution obtaining, then water supplements and reaches cumulative volume 1000ml again; Finally, use mineral acid or mineral alkali that the pH value of solution is adjusted to 6.8～7.0.
5. cultural method according to claim 2, is characterized in that the preparation method of described fermention medium is as follows:
1～10 gram of yeast extract paste, 5～20 grams of glycerine, 0.1～2 gram of saltpetre, 0.1～2 gram of Secondary ammonium phosphate, 0.1～1 gram of manganous sulfate, 0.1～1 gram of iron trichloride, 0.1～2 gram of dipotassium hydrogen phosphate, 0.1～2 gram of potassium primary phosphate and 0.1～1 gram of sodium-chlor is soluble in water, and then water supplements and reaches cumulative volume 1000ml; Finally, use mineral acid or mineral alkali that the pH value of solution is adjusted to 6.8～7.0.
6. according to the cultural method described in any one claim in claim 3-5, it is characterized in that described mineral acid is selected from sulfuric acid or hydrochloric acid; Described mineral alkali is selected from sodium hydroxide or potassium hydroxide.
7. according to the cultural method described in any one claim in claim 3-5, the concentration that it is characterized in that described mineral acid or inorganic base aqueous solution is 5-6M.
8. the resulting Bradyrhizobium sp Arachis agent of cultural method according to claim 2.
9. Bradyrhizobium sp Arachis agent according to claim 8 purposes in peanut cultivation.
10. purposes according to claim 9, is characterized in that in the peanut cultivation phase, and described Bradyrhizobium sp Arachis agent, mixes seed dressing by the consumption on 140～160ml/ mu of ground and peanut seed, then sowing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310643826.5A CN103627662B (en) | 2013-12-03 | 2013-12-03 | A kind of Bradyrhizobium sp Arachis and uses thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310643826.5A CN103627662B (en) | 2013-12-03 | 2013-12-03 | A kind of Bradyrhizobium sp Arachis and uses thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103627662A true CN103627662A (en) | 2014-03-12 |
| CN103627662B CN103627662B (en) | 2015-09-16 |
Family
ID=50209114
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310643826.5A Active CN103627662B (en) | 2013-12-03 | 2013-12-03 | A kind of Bradyrhizobium sp Arachis and uses thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103627662B (en) |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104396592A (en) * | 2014-12-11 | 2015-03-11 | 东北农业大学 | Novel synchronous detection method of soil rhizobium japonicum separation and nitrogen fixation activity |
| CN104705347A (en) * | 2015-02-12 | 2015-06-17 | 兴邦（武汉）生物科技有限公司 | Peanut slow-growing nitragin as well as preparation method and application thereof |
| CN105176878A (en) * | 2015-10-13 | 2015-12-23 | 王�琦 | Peanut rhizobium culture medium and method for preparing peanut rhizobium inoculant by adopting peanut rhizobium culture medium |
| CN105567615A (en) * | 2016-03-14 | 2016-05-11 | 中国科学院烟台海岸带研究所 | Bradyrhizobium sp. and application thereof |
| CN105950520A (en) * | 2016-07-18 | 2016-09-21 | 武汉市农业科学技术研究院作物科学研究所 | Rhizobium capable of efficiently solubilizing phosphorus and application of rhizobium |
| CN109294962A (en) * | 2018-12-05 | 2019-02-01 | 福建农林大学 | One plant of Cassia rhizobium TXN1 and its application |
| CN109337847A (en) * | 2018-12-05 | 2019-02-15 | 福建农林大学 | One plant of Cassia rhizobium TXR2 and its application |
| CN109355235A (en) * | 2018-12-05 | 2019-02-19 | 福建农林大学 | A kind of rhizobium FAMB126 and its application |
| CN109355234A (en) * | 2018-12-05 | 2019-02-19 | 福建农林大学 | A kind of rhizobium YZM0144 and its application |
| CN109439589A (en) * | 2018-12-05 | 2019-03-08 | 福建农林大学 | A kind of rhizobium YZLH133 and its application |
| CN109486711A (en) * | 2018-12-05 | 2019-03-19 | 福建农林大学 | A kind of rhizobium AXLQ16 and its application |
| CN115197879A (en) * | 2022-06-29 | 2022-10-18 | 华南农业大学 | Slow rhizobium W052 and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643761A (en) * | 2011-10-08 | 2012-08-22 | 中国农业科学院农业资源与农业区划研究所 | Rhizobium with rice growth promoting function and application thereof |
-
2013
- 2013-12-03 CN CN201310643826.5A patent/CN103627662B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643761A (en) * | 2011-10-08 | 2012-08-22 | 中国农业科学院农业资源与农业区划研究所 | Rhizobium with rice growth promoting function and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| 吴海燕，等: "花生根瘤菌高效菌株的筛选及固氮效应的研究", 《吉林农业科学》, vol. 28, no. 6, 31 December 2003 (2003-12-31), pages 20 - 25 * |
| 朱剑光，等: "花生慢生根瘤菌的分离与鉴定", 《生物技术》, vol. 16, no. 2, 30 April 2006 (2006-04-30), pages 45 - 47 * |
| 杨庆锋 等: "花生根瘤菌的应用效果研究", 《花生学报》, vol. 42, no. 3, 15 September 2013 (2013-09-15), pages 56 - 59 * |
| 韩梅 等: "一种高效花生根瘤菌的筛选与鉴定", 《微生物学杂志》, vol. 33, no. 1, 28 February 2013 (2013-02-28), pages 44 - 47 * |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104396592A (en) * | 2014-12-11 | 2015-03-11 | 东北农业大学 | Novel synchronous detection method of soil rhizobium japonicum separation and nitrogen fixation activity |
| CN104705347A (en) * | 2015-02-12 | 2015-06-17 | 兴邦（武汉）生物科技有限公司 | Peanut slow-growing nitragin as well as preparation method and application thereof |
| CN104705347B (en) * | 2015-02-12 | 2018-03-27 | 兴邦（武汉）生物科技有限公司 | One cultivates peanut Slow_growing rhizobia agent, preparation method and use |
| CN105176878A (en) * | 2015-10-13 | 2015-12-23 | 王�琦 | Peanut rhizobium culture medium and method for preparing peanut rhizobium inoculant by adopting peanut rhizobium culture medium |
| CN105567615A (en) * | 2016-03-14 | 2016-05-11 | 中国科学院烟台海岸带研究所 | Bradyrhizobium sp. and application thereof |
| CN105950520B (en) * | 2016-07-18 | 2019-05-10 | 武汉市农业科学技术研究院作物科学研究所 | A kind of rhizobium of efficient phosphate-solubilizing and its application |
| CN105950520A (en) * | 2016-07-18 | 2016-09-21 | 武汉市农业科学技术研究院作物科学研究所 | Rhizobium capable of efficiently solubilizing phosphorus and application of rhizobium |
| CN109294962A (en) * | 2018-12-05 | 2019-02-01 | 福建农林大学 | One plant of Cassia rhizobium TXN1 and its application |
| CN109355235A (en) * | 2018-12-05 | 2019-02-19 | 福建农林大学 | A kind of rhizobium FAMB126 and its application |
| CN109355234A (en) * | 2018-12-05 | 2019-02-19 | 福建农林大学 | A kind of rhizobium YZM0144 and its application |
| CN109439589A (en) * | 2018-12-05 | 2019-03-08 | 福建农林大学 | A kind of rhizobium YZLH133 and its application |
| CN109486711A (en) * | 2018-12-05 | 2019-03-19 | 福建农林大学 | A kind of rhizobium AXLQ16 and its application |
| CN109337847A (en) * | 2018-12-05 | 2019-02-15 | 福建农林大学 | One plant of Cassia rhizobium TXR2 and its application |
| CN115197879A (en) * | 2022-06-29 | 2022-10-18 | 华南农业大学 | Slow rhizobium W052 and application thereof |
| CN115197879B (en) * | 2022-06-29 | 2023-08-22 | 华南农业大学 | Rhizobium chromenensis W052 and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103627662B (en) | 2015-09-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103627662B (en) | A kind of Bradyrhizobium sp Arachis and uses thereof | |
| CN110438037B (en) | Klebsiella sp 5 with phosphorus dissolving effect and application thereof | |
| CN102876608B (en) | Bacillus amyloliquefaciens and application thereof | |
| CN103320360B (en) | Bacillus megatherium SJ-7 strain and application thereof | |
| CN104928212A (en) | Bacillus megaterium strain X3 and preparation method and application thereof | |
| CN103275895B (en) | Saline-alkali-tolerant heteroauxin-producing Bacillus subtilis and application thereof | |
| CN106967652B (en) | Rhizobium for promoting growth of common vetch and application of rhizobium | |
| CN110577911B (en) | Bacillus pumilus and application thereof | |
| CN103571770B (en) | A kind of Efficient peanut rhizobiumleguminosarstrain strain and application thereof | |
| CN105274030A (en) | Rhizobium and application thereof | |
| CN103224897A (en) | Bacillussubtilis for tobacco black shank prevention and control | |
| CN114908014B (en) | Tea-oil tree endophyte capable of promoting dissolution of ferric phosphate and application of tea-oil tree endophyte | |
| CN109182194B (en) | Rhizobium oridonii for promoting growth of corolla dentiger and culture method and application thereof | |
| CN109055274B (en) | Caragana rhizobium and fermentation culture method and application thereof | |
| CN107628894A (en) | Composite bacteria agent increase soil fertility and its preparation method and application | |
| CN104560817B (en) | Thermophilic bacillus licheniformis UTM102 for producing phytase and application of thermophilic bacillus licheniformis UTM102 | |
| CN103952332A (en) | Bacillus amyloliquefaciens ZH1 and its application | |
| CN101955903A (en) | Bacillus licheniformis bacterial strain and application thereof | |
| CN105296392A (en) | Fermentation method of bacillus amyloliquefaciens Bam22 for preventing and treating cruciferae plasmodiophora brassicae | |
| CN101914468A (en) | Nitrogen-fixing bacillus megaterium strain DL7 and application thereof | |
| CN1323161C (en) | Lucerne rhizobium and its fermentation culture method and uses | |
| CN103045500B (en) | Mesorhizobium KDRM295 and application thereof | |
| CN109609412A (en) | A kind of Thermophilic Bacteria Bacillus smithii Ths1 and its application | |
| CN110438026B (en) | Bacillus amyloliquefaciens GLD-191 and microbial inoculum, preparation method and application thereof | |
| CN108841761B (en) | Method for promoting growth of clover and/or increasing yield of clover and microbial inoculum used by method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | 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 |