CN105907670B - A kind of preparation method and application of microbacterium and its bacteria agent for cassava fixed nitrogen, the bacteria agent - Google Patents
A kind of preparation method and application of microbacterium and its bacteria agent for cassava fixed nitrogen, the bacteria agent Download PDFInfo
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Abstract
The invention discloses the microbacteriums and preparation method and application for cassava fixed nitrogen, are Microbacterium (Microbacterium sp.).Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation day: on 03 07th, 2016, deposit number: CGMCC No.12182；Using the microbacterium as the bacteria agent of active constituent, by it after the culture of pipe kind, liquid fermentation and culture manufactured bacteria agent.The nitrogen content that cassava seedling root, stem, leaf can be significantly improved using the strain and its bacteria agent facilitates the nitrogen accumulation that cassava grows early period, has preferable application value in the production of cassava.
Description
Technical field
The invention belongs to the nitrogen-fixing bacteria field in microorganisms technical field, in particular to a kind of microbot for cassava fixed nitrogen
The preparation method and application of bacterium and its bacteria agent, the bacteria agent.
Background technique
Endophytic Diazotroph, which refers to, to be colonized in plant, and the micro- life of one kind of association nitrogen fixation can be carried out with host plant
Object.The nitrogen of free state in atmosphere can be transformed into the nitrogen that plant can directly be absorbed and utilized using interior azotobacter, then into
The macromolecular substances such as protein needed for one step is transformed into plant, nucleic acid supply needed for plant growth.From agriculturally, utilize
Biological nitrogen fixation is that one of the important channel of nitrogenous fertilizer is provided for crop.Since the 1980s, interior azotobacter is due in grass
There is nitrogenase activity on section crop, and plant growth can be promoted, become the hot spot studied both at home and abroad.
The mechanism of association nitrogen fixation: since 1972, Brazilian scientistIt is sent out when studying gramineae plant
Show interior azotobacter, has attracted extensive attention, and formed in research herbages in 1974 and azospirillum (Azospirillum)
In Nitrogen Fixation System, it is believed that azotobacter strain can invade in the cortical tissue or dimension pipe of host organ, but not form symbiotic tissues,
They are in the commensalism of a kind of " loose " with host cell, referred to as " united symbiosis nitrogen fixation "
(associativesymbioticnitrogenfixation).There are two the mechanism of combination azotobacter promotion plant growth is main
Aspect, wherein being to provide nitrogen for plant by biological nitrogen fixation or generate plant hormone to directly facilitate plant growth, the second is logical
It crosses induction plant to generate plant hormone, improve plant to the absorption of mineral element and utilize and promote plant growth indirectly, promotion
Root system development.
In recent years, researcher both domestic and external is successively separated in a variety of from the gramineae plants such as sugarcane, rice, rye grass
Azotobacter, in addition, also having also discovered interior azotobacter from Lin Benzhong such as palm, fruit tree, coffee tree, black pines.In recent years
Interior azotobacter reported in the literature has: fixed nitrogen acetobacter (Gluconacetobacterdiazotrophicus), Sai Lupuka grass
Spirillum (Herbaspirillumseropedicae) and fixed nitrogen vibrios (Azoarus), bulkholderia cepasea
(Burkholderia), azospirillum (Azospirillum), Klebsiella (Klebsiella), bacillus
(Bacillus), enterobacteria (Enterobacter), pseudomonad (Pseudomonas), nitrogen-fixing rhizobia
(Azorhizobium) etc. some kinds in belonging to.Corn, rice, wheat Field inoculation combination azotobacter experimental result table
It is bright, 10% or so can be increased production using corresponding Associative nitrogen-fixing bacteria microbial inoculum.But up to the present, it is not yet found that closing in cassava
The report of interior azotobacter.
The application of nitrogenous fertilizer plays irreplaceable role to the development of modern agriculture.The nitrogen fertilizer production amount in China and consumption
Amount occupies first place in the world.But China's agricultural necessarily causes the risk of diminishing returns and environmental pollution to the excessive dependence of nitrogenous fertilizer,
Acidification, salination, water eutrophication and the Global Greenhouse Effect of such as soil.Nitrogen, biological nitrogen fixation tool are applied compared to chemistry
Have the advantages that economical and environmentally friendly, not only to reduce agricultural cost, but also avoids chemistry and apply various environmental problems brought by nitrogen.It is interior
Azotobacter can promote cassava growth with cassava association nitrogen fixation after disseminating cassava, reduce nitrogenous fertilizer and use.Separation and screening are with wood
Potato is the interior azotobacter of host, and promoting cassava growth using it is the purpose of the present invention.
Summary of the invention
To solve the above-mentioned problems of the prior art, the first object of the present invention is to provide a kind of for cassava fixed nitrogen
Microbacterium, second is designed to provide the bacteria agent and preparation method thereof using the bacterial strain as active constituent, third mesh
Be the application of the bacterial strain and its bacteria agent is provided.
A kind of preparation method of the microbacterium for cassava fixed nitrogen, step are as follows:
Step 1: sample is chosen: being sampled to cassava, choose the part that the root of cassava is not expanded, choose a fixed length
The complete root of degree, diameter 2-3mm；
Step 2: the cassava root of acquisition is classified according to root long, being roughly divided into 4 classes is respectively 5cm, 10cm, 15cm,
20cm is clean with distilled water flushing；The manioc root classified is subjected to surface disinfection, the manioc root nothing that surface disinfection is completed
Bacterium water rinses 5 times, last is coated on LB culture medium all over the sterile water rinsed, and 37 DEG C of culture 36h determine that sterile water is coated with
Culture medium on without thalli growth, it was demonstrated that sufficiently external source thallus is completely not present in sterilizing for root outer surface, then by root material
Screening for endophyte；
Step 3: sterilizing after step 2 is handled and the root rinsed well is put into mortar and grinds, the sterile of 10mL is added
5min is stood after water, and the lapping liquid of 0.1mL is taken to be coated on nitrogen-free solid medium, 28 DEG C of culture 5-7d；Observe nitrogen-free solid
Bacterial growth situation on culture medium chooses single bacterium colony that is can growing and can provoking and carries out scribing line culture；In nitrogen-free
Carry out scribing line culture on solid medium, passage choose afterwards three times can secondary culture, and the bacterium for the separation that can cross into
Row follow-up test；
Step 4: carrying out Genome DNA extraction to growing nitrogen-fixing bacterial strain in gained, and using total DNA as template, 16SrRNA base is expanded
Because as a result sequence is compared after amplified production sequencing on NCBI.
Further, in the step 1, seedling stage cassava is chosen in sampling.
Further, in the step 1, the length of cassava root is chosen in 5cm or more.
Further, in the step 3, the composition of nitrogen-free solid medium are as follows: sucrose 10g, K2HPO4·H2O0.1g,
MgSO4·H2O0.2g, CaCl2·H2O0.02g, Na2MoO4·H2O0.002g, Malicacid5.0g, KH2PO4·H2O0.4g,
NaCl0.1g, FeCl30.01g, distilled water 1000mL, pH7.0.
The cassava diazotroph microbial inoculum as made from above-mentioned microbacterium, the bacteria agent is by the microbacterium through pipe kind culture
Or it is made after liquid fermentation and culture；Bacterial concentration in bacteria agent are as follows: Shuo≤10 Jun living every mL8。
Further, the preparation method of the bacteria agent, is respectively as follows:
A, pipe kind culture: by strain inoculated to test tube slant culture medium, constant temperature incubation 2-3 days at 28-32 DEG C of temperature,
It can be obtained；
B: liquid fermentation: test tube species are inoculated on fluid nutrient medium, 28-32 DEG C shaking table culture 1-2 days, mesh can be obtained
Mark the bacteria agent that strain is active constituent.
Further, the ingredient of test tube slant culture medium described in the culture of A pipe kind are as follows: peptone 10g, yeast extract
5g, NaCl10g, distilled water 1000mL, agar 15-20g, pH7.0-7.2；
Liquid Culture based component described in B liquid fermentation are as follows: peptone 10g, yeast extract 5g, NaCl10g, distillation
Water 1000mL, pH7.0-7.2.
Microbacterium and its bacteria agent for cassava fixed nitrogen are improving the application in cassava seedling stage nitrogen accumulation.
Compared with the existing technology, the invention has the benefit that the obtained bacterial strain of the present invention and its biological bacteria of development
Agent can effectively improve the accumulation of cassava seedling stage nitrogen.Nitrogenase activity is 37.77nmolmL-1·h-1。
Detailed description of the invention
Fig. 1 is that bacterial strain of the present invention grows figure on nitrogen-free agar.
Fig. 2 is bacterial strain Gram's staining figure of the present invention.
Specific embodiment
Technical solution of the present invention is described in further detail With reference to embodiment:
Test example:
Azotobacter A08 bacterial strain, belongs to Firmicutes (Firmicutes out of be separated in cassava 1 plant；) microbacterium
Belong to (Microbacteriumsp.).Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center
(CGMCC), preservation day: on 03 07th, 2016, deposit number: 12182；
The preparation method of the interior azotobacter being separated to from cassava includes the following steps:
A. isolation medium:
Dobereiner nitrogen-free agar: sucrose 10g, K2HPO4·H2O0.1g, MgSO4·H2O0.2g, CaCl2·
H2O0.02g, Na2MoO4·H2O0.002g, Malicacid5.0g, KH2PO4·H2O0.4g, NaCl0.1g, FeCl30.01g steams
Distilled water 1000mL, pH7.0.
B. cultural method:
(1) sample is chosen: being sampled in the seedling stage of cassava, chooses the part that the root of cassava is not expanded, choose as far as possible
Complete root, for length in 5cm or more, diameter is 2-3mm or so.
(2) specific steps: the cassava root of acquisition is classified according to root long, be roughly divided into 4 classes be respectively 5cm, 10cm,
15cm and 20cm is clean with distilled water flushing.The manioc root classified is subjected to surface disinfection, specific sterilizing time processing and knot
Fruit is shown in Table 1.The manioc root that surface disinfection is completed is coated on LB all over the sterile water rinsed with aseptic water washing 5 times, by last
On culture medium, 37 DEG C of culture 36h have seen whether bacterium growth, indicate that surface disinfection is incomplete if there is thallus is grown, material is not
It can be used for the screening of endophyte；If growing without thallus indicates that surface disinfection is complete, material can be used for the screening of endophyte.It will
It sterilizes and the root rinsed well is put into mortar and grinds, stand 5min after the sterile water of 10mL is added, the lapping liquid of 0.1mL is taken to apply
Cloth is on nitrogen-free solid medium, 28 DEG C of culture 5-7d.The bacterial growth situation on nitrogen-free solid medium is observed, selection can
Single bacterium colony that is growing and can provoking carries out scribing line culture.Scribing line culture, passage three are carried out on nitrogen-free solid medium
It is secondary after choose can secondary culture, and the separation that can cross bacterium carry out follow-up test.
The processing of 1 cassava root surface disinfection of table and result
Note: "-" indicates have bacterium colony to grow in this culture medium, and "+" indicates do not have bacterium colony to grow in this culture medium.
(3) phase, root tuber expanding stage are formed in the root tuber of cassava, the root tuber maturity period carries out bolter to the root sampling of cassava
The experiment of azotobacter is failed in selecting.
The separated bacterium arrived of above-mentioned steps is measured into nitrogen fixation enzymatic activity by acetylene reduction method, bacterium is identified with this
Whether there is the ability of biological nitrogen fixation.The specific method is as follows:
The endophyte isolated is inoculated in the 100mL triangular flask equipped with the bottom 20mL nitrogen Dobereiner fluid nutrient medium
In, cover tampon.30 DEG C, 150r/min culture.Tampon is changed into rubber stopper when the OD600 of bacterium solution reaches 0.6, uses syringe
It extracts gas 5mL in bottle out, injects high purity acetylene 5mL, continue culture for 24 hours.With the production quantity of gas chromatograph for determination ethylene, press
Formula calculates nitrogenase activity.
C=(hx × c × V)/(24.9 × hs × t)
Wherein, hx is sample peak area value；
Hs is standard C2H4Peak area value；
C is standard C2H4Concentration (nmol/mL)；
V is culture vessel volume (mL)；
T is the sample culturing time (h)；
C is the C generated2H4Concentration (nmolmL-1·h-1)。
Isolation and screening of bacterial strain result:
1 plant of bacterial strain with higher nitrogenase activity is obtained by screening to azotobacter in cassava root and identification,
For A08 bacterial strain.Its nitrogenase activity is 37.77nmolmL-1·h-1.By the strain inoculated into cassava, in potted plant experiment
As a result the nitrogen content of cassava seedling stage overground part and underground part can be improved in.
Bacterial strain identification method:
Cultural characteristic and the Physiology and biochemistry identification of bacterial strain are referring to " common bacteria system identification handbook " and " Weberian identification hand
Volume ".The Genome DNA extraction of bacterial strain uses kit, and using total DNA as template, expands 16SrRNA gene order, and amplified production is sent
To Nanning, Guo Tuo biotech firm is sequenced, and is as a result compared on NCBI.
Qualification result:
The first purpose of this invention, obtained strains: A08 bacterial strain is that separation screens from cassava seedling stage root, is passed through
Bio-chemical characteristics and 16SrRNA Sequence Identification determine that A08 bacterial strain belongs to Firmicutes (Firmicutes；) Microbacterium
(Microbacterium)。
Bacterial strain main feature are as follows:
As shown in Figure 1, 2, A08 bacterium colony is creamy white circle on LB culture medium, surface wettability protrusion, neat in edge.Leather
Lan Shi is positive, and Starch Hydrolysis, gelatin liquefaction, lactose fermentation are feminine gender.Methyl red, indoles experiment, V.P, citrate, sucrose
Fermentation, maltose fermentation, glucose fermentation are the positive.
Second object of the present invention is achieved in that be made after the culture of pipe kind and liquid fermentation with above-mentioned bacterial strains
At bacteria agent.
The preparation method of above-mentioned bacteria agent specifically: including the culture of pipe kind and liquid fermentation experimental method.It specifically includes:
A: pipe kind culture: A08 being inoculated into respectively on two test tube slant culture mediums, and constant temperature is trained at 28-32 DEG C of temperature
It supports 2-3 days, can be obtained.
B: liquid fermentation: test tube species are inoculated on fluid nutrient medium, 28-32 DEG C shaking table culture 1-2 days, mesh can be obtained
Mark the bacteria agent that strain is active constituent.
The bacteria agent for being achieved in that the present invention develops of third object of the present invention is remarkably improved potting
Cassava seedling stage nitrogen accumulation amount.
The present invention filters out the bacterial strain with higher nitrogenase activity from cassava root, and develops effective biological bacteria
Agent, the bacteria agent can effectively improve potting cassava seedling stage nitrogen accumulation.
Experimental example 2
The present invention is that the interior azotobacter A08 bacterial strain belongs to Firmicutes (Firmicutes；) Microbacterium
(Microbacteriumsp.).Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center
(CGMCC), preservation day: on 03 07th, 2016, deposit number: 12182；
Microbacterium of the present invention is the bacteria agent of active constituent, is with above-mentioned bacterial strains by the culture of pipe kind and liquid
Manufactured bacteria agent after fermentation.
The preparation method of bacteria agent of the present invention, including the culture of pipe kind and liquid fermentation experimental method.Specific packet
It includes:
A: pipe kind culture: A08 is inoculated on test tube slant culture medium, constant temperature incubation 2-3 days at 28-32 DEG C of temperature,
It can be obtained.
B: liquid fermentation: test tube species are inoculated on fluid nutrient medium, 28-32 DEG C shaking table culture 1-2 days, mesh can be obtained
Mark the bacteria agent that strain is active constituent.
Tube propagation based component used in step A are as follows: peptone 10g, yeast extract 5g, NaCl10g, distilled water
1000mL, agar 15-20g, pH7.0-7.2.
Liquid fermentation medium ingredient used in step B are as follows: peptone 10g, yeast extract 5g, NaCl10g, distilled water
1000mL, pH7.0-7.2.
Shaking speed used in step B is 150-180rmp.
The preparation concrete operations of bacteria agent of the present invention are as follows:
A: the culture medium that pipe kind culture uses are as follows: peptone 10g, yeast extract 5g, NaCl10g, distilled water 1000mL,
Agar 15-20g, pH7.0-7.2.
A08 is inoculated on test tube slant culture medium, constant temperature incubation 2-3 days, can be obtained at 28-32 DEG C of temperature.
B: liquid fermentation medium ingredient used in liquid fermentation are as follows: peptone 10g, yeast extract 5g, NaCl10g steam
Distilled water 1000mL, pH7.0-7.2.
Test tube species are inoculated into 500mL triangular flask, have 200mL fluid nutrient medium per bottled, in 28-32 DEG C of constant temperature
Under, 150-180rmp shaking table culture 1-2 days detects the concentration of bacteria suspension with OD600, when OD600 is between 0.4-0.8, carefully
Bacterium is in logarithmic growth phase, viable count about 108-1010, effective bacteria agent as obtained.
The preparation result of bacteria agent:
Pass through the concentration for the bacteria agent that liquid fermentation obtains are as follows: Shuo≤10 Jun living every mL8。
The application of bacteria agent:
Influence experiment of the bacteria agent of the present invention to cassava seedling stage nitrogen accumulation
(1) it prepares bacteria agent: getting out bacteria agent according to the preparation method of the bacteria agent of above-mentioned A08 bacterial strain, with
The fermentation liquid of bacterium is not connect as control.
(2) cassava nursery: cassava seed stems being intercepted as 2-3cm size, nursery is carried out in perlite, when cassava seedling is long extremely
It is transplanted when 2-3 leaf.
(3) potting prepares: perlite and sand are distinguished into autoclave sterilization, with perlite and soil ratio for 3:
It is fitted into basin after 1 mixing, the specification of basin used is internal diameter 21-23cm, the opaque flowerpot of high 30cm.Mixing is packed into every basin
Native 4kg.
(4) nutrient solution prepares: nutrient solution used is complete Huo Gelan nutrient solution and nitrogen-free Glan nutrient solution suddenly.
(5) processing method of the bacteria agent to cassava seedling: the cassava seedling educated in perlite to 2-3 leaf is carefully taken out,
Its root must not be hurt.With aseptic water washing after clean by the root of cassava seedling in order to achieve the above objectives, technical solution of the present invention
Are as follows:
A kind of microbacterium for cassava fixed nitrogen its for it be Firmicutes Firmicutes；Microbacterium
Microbacteriumsp., deposit number are as follows: CGMCCNo.12182.Deposit number are as follows: CGMCCNo.12182.
Further, the microbacterium is in yellow on LB culture medium, round, and surface wettability protrusion, neat in edge is easily chosen
It rises；Gram positive bacterial strain, indoles experiment, diacetyl test, glucose fermentation are the positive, and methyl red, Starch Hydrolysis are real
It tests, gelatin liquefaction experiment, citrate experiment, lactose fermentation, sucrose ferments, maltose fermentation is feminine gender.
It is immersed in bacteria agent and is transplanted into basin after 30min.Control is immersed in the fermentation liquid not being inoculated with.
3 groups of experiment point: inoculation A08 bacteria agent is not inoculated with CK1, is not inoculated with CK2.Every group 5 parallel.
(6) potting cassava growth period is handled: applying nutrient solution 100mL after transplanting into flowerpot every 2 days.Until 30 days
Experiment terminates afterwards.
It applies situation and is respectively as follows: A08 processing group and CK1 application nitrogen-free Glan nutrient solution suddenly.CK2 applies complete Huo Gelan battalion
Nutrient solution.
(7) to the root of cassava and the nitrogenase activity detection method of blade after endophyte dip dyeing cassava
Receipts sample, positive 3 leaves of acquisition cassava and root are carried out after experiment process 30 days, aseptic water washing is clean, with 0.1%
Mercuric chloride is used aseptic water washing 5 times after impregnating 10min, respectively shreds root, stem and leaf in the vial for being fitted into 50mL, is had per bottled
The nitrogen-free agar of 10mL, rubber stopper sealing are injected 1mL high purity acetylene after extracting 1mL air out with syringe, are extracted after cultivating 2h
1mL gas carries out the production quantity of gas chromatographic detection ethylene, carries out calculating nitrogenase activity with following formula.
C=(hx × c × V)/(24.9 × hs × t)
Wherein, hx is sample peak area value；
Hs is standard C2H4Peak area value；
C is standard C2H4Concentration (nmol/mL)；
V is culture vessel volume (mL)；
T is the sample culturing time (h)；
C is the C generated2H4Concentration (nmolmL-1·h-1)。
(8) the nitrogenase activity testing result of manioc root, leaf
By the nitrogenase activity testing result table 2 of root, leaf to the cassava for disseminating bacteria agent it is found that in root and leaf
Piece detects nitrogenase activity, shows that bacteria agent can disseminate cassava, and play nitrogen fixation in root and blade.
Nitrogenase activity (the nmolmL of 2 manioc root of table, leaf-1·h-1)
(9) endophyte disseminates the analysis method of cassava seedling stage nitrogen content after cassava
Carry out receipts sample after experiment process 30 days, by the root, stem, leaf of cassava adopt respectively it is lower rinsed well with deionized water after point
It is not fitted into envelope and dries in an oven, wherein nitrogen content is measured after crushing.The measurement of nitrogen content is disappeared using sulfuric acid boils-Na Shi ratio
Color method.
(10) application effect of bacteria agent
Two kinds of bacteria agents that the present invention obtains can be disseminated by cassava root and be played on root and leaf solid
Nitrogen effect, to reach the increased effect of cassava seedling nitrogen.From table 3 it is recognised that using bacteria agent to the root of cassava,
Stem, leaf nitrogen content be significantly increased.Using the blade of the cassava of A08 bacteria agent, stem and root nitrogen content and do not apply nitrogen,
The CK1 that bacteria agent is not added is compared, and is increased significantly.Compared with normally applying nitrogen, the CK2 of bacteria agent is not added, A08 is used
Blade, stem and the nitrogen content of root of the cassava of bacteria agent respectively reach the 87.51%, 85.29% and 80.35% of CK2,
Illustrate the part amount of nitrogen that cassava seedling stage can be substituted using A08 bacteria agent.
The nitrogen content of the root of 3 cassava of table, stem, leaf
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any
The change or replacement expected without creative work, should be covered by the protection scope of the present invention.Therefore, of the invention
Protection scope should be determined by the scope of protection defined in the claims.
Claims (6)
1. a kind of microbacterium for cassava fixed nitrogen, which is characterized in that it is Firmicutes Firmicutes；Microbacterium
Microbacterium sp., deposit number are as follows: CGMCC No.12182.
2. a kind of microbacterium for cassava fixed nitrogen according to claim 1, which is characterized in that the microbacterium is trained in LB
Supporting is in yellow on base, round, and surface wettability protrusion, neat in edge is easily provoked；Gram positive bacterial strain, indoles experiment, diethyl
Acyl test, glucose fermentation are the positive, methyl red, Starch Hydrolysis experiment, gelatin liquefaction experiment, citrate experiment, lactose
Fermentation, sucrose fermentation, maltose fermentation are feminine gender.
3. the cassava diazotroph microbial inoculum as made from microbacterium described in claim 1, which is characterized in that the bacteria agent is by institute
Microbacterium is stated to be made after the culture of pipe kind or liquid fermentation and culture；Bacterial concentration in bacteria agent are as follows: every mL Jun Shuo living≤
108。
4. the preparation method of bacteria agent as claimed in claim 3, which is characterized in that be respectively as follows: A, pipe kind culture: bacterial strain is connect
In kind to test tube slant culture medium, constant temperature incubation 2-3 days, be can be obtained at 28-32 DEG C of temperature；
B: liquid fermentation: test tube species are inoculated on fluid nutrient medium, 28-32 DEG C shaking table culture 1-2 days, object bacteria can be obtained
Kind is the bacteria agent of active constituent.
5. the preparation method of bacteria agent according to claim 4, it is characterised in that test tube described in A pipe kind culture is oblique
The ingredient of face culture medium are as follows: peptone 10g, yeast extract 5g, NaCl 10g, distilled water 1000mL, agar 15-20g,
pH7.0-7.2；
Liquid Culture based component described in B liquid fermentation are as follows: peptone 10g, yeast extract 5g, NaCl 10g, distilled water
1000mL, pH7.0-7.2.
6. described in claim 3 for the microbacterium of cassava fixed nitrogen and its bacteria agent in improving cassava seedling stage nitrogen accumulation
Using.
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| WO2013141815A1 (en) * | 2012-03-21 | 2013-09-26 | Temasek Life Sciences Laboratory Limited | Nitrogen-fixing bacterial inoculant for improvement of crop productivity and reduction of nitrous oxide emission |
| CN105237075A (en) * | 2015-09-28 | 2016-01-13 | 广西洪喜肥业有限公司 | Special manihot esculenta crantz compound fertilizer and production method thereof |
Non-Patent Citations (3)
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| Érica. L. Reinhardt等.Molecular characterization of nitrogen-fixing bacteria isolated from brazilian agricultural plants at São Paulo state.《Brazilian Journal of Microbiology》.2008,第39卷(第3期),414-422. * |
| 玉米内生固氮菌的分离鉴定及其促生长作用研究_韩梅;韩梅等;《沈阳农业大学学报》;20100228;第41卷(第1期);94-97 * |
| 自生固氮菌的分离鉴定;杨从发等;《淮海红学院学报》;19990930;第8卷(第3期);56-58 * |
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