CN109880759A - A kind of rihizobium japonicum for being suitble to that freeze dried powder is made and its application - Google Patents
A kind of rihizobium japonicum for being suitble to that freeze dried powder is made and its application Download PDFInfo
- Publication number
- CN109880759A CN109880759A CN201910168059.4A CN201910168059A CN109880759A CN 109880759 A CN109880759 A CN 109880759A CN 201910168059 A CN201910168059 A CN 201910168059A CN 109880759 A CN109880759 A CN 109880759A
- Authority
- CN
- China
- Prior art keywords
- dried powder
- freeze
- slow
- japonicum
- freeze dried
- 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
A kind of rihizobium japonicum for being suitble to that freeze dried powder is made and its application, it is related to a kind of Slow-growing Soybean rhizobia and its application.The present invention is poor in order to solve the problems, such as existing rhizobium resistance to low temperature.The rihizobium japonicum that the present invention is suitble to that freeze dried powder is made is Slow-growing Soybean rhizobia (Bradyrhizobium japonicum) Bw 0122, can be used as freeze dried powder and is applied.Number of viable has excellent freeze drying activity without being decreased obviously after 0122 freezing processing of Slow-growing Soybean rhizobia Bw of the present invention.
Description
Technical field
The present invention relates to a kind of Slow-growing Soybean rhizobia and its applications.
Background technique
Molecular nitrogen in air can be changed into ammoniacal nitrogen by the symbiotic azotification of rhizobium by leguminous plant,
It supplies plant to use, a small root nodule is exactly the nitrogen fertilizer plant of a seat minitype.Production can be improved in legume crops Rhizobium Inoculation
Amount, especially soybean, peanut and other crops Rhizobium Inoculation technology oneself become agricultural production routine techniques.Nitragin
Using quantity, quality and the amount of nitrogen fixation for being conducive to improve legume nodule, the use of chemical fertilizer can be effectively reduced, improves soil
And eco-environmental quality, crop yield and quality are improved, the adjustment of agricultural structure is conducive to.In short, rhizobium are ground
Study carefully and applies and will improve crop yield, reduction fertilizer amount and production cost, reduction water and soil pollution and soil-borne disease, build
Vertical ecological balance etc. plays a significant role.Rhizobium generally use freeze dried powder to carry out storage and transport, but existing
Tumor bacterium resistance to low temperature is poor, and after lyophilized technique is handled, the quantity of viable bacteria body often declines obviously, influences its biological effect
Performance.
Freeze dried powder is also usually made in rhizobium, for dressing seed, to improve the yield of the crops such as soybean, because it has easily
In the storage and transport the advantages that, there is vast potential for future development, however existing rhizobium resistance to low temperature is poor, by freeze-drying
The decline of living bacteria count amount is obvious after process, limits its use.
Summary of the invention
The present invention is poor in order to solve the problems, such as existing rhizobium resistance to low temperature, and provides one kind and be suitble to that freeze-dried powder is made
The rihizobium japonicum of agent and its application.
The present invention is suitble to that the rihizobium japonicum of freeze dried powder is made, and is Slow-growing Soybean rhizobia
(Bradyrhizobium japonicum) Bw 0122 is preserved in China Committee for Culture Collection of Microorganisms's commonly micro- life
Object center, deposit number are CGMCC No.16010.
Slow-growing Soybean rhizobia (Bradyrhizobium japonicum) Bw 0122 of the present invention needs for Gram-negative
Oxygen bacillus, 0.5~0.9 1.2~6.0 μm of μ m.
0122 bacterium colony circular protrusions of Slow-growing Soybean rhizobia (Bradyrhizobium japonicum) Bw of the present invention,
White, opaque, surface is smooth, and edge is irregular.
The present invention uses above-mentioned rihizobium japonicum as the application of freeze dried powder.Slow-growing Soybean rhizobia of the present invention
Rihizobium japonicum freeze dried powder is made by frozen dried in (Bradyrhizobium japonicum) Bw 0122, after freeze-drying
Living bacteria count is compared before living bacteria count and freeze-drying, and living bacteria count amount reaches 92% or more after freeze-drying；Soybean nodulation
Bacterium freeze dried powder is after 10 DEG C of storage 90d, and living bacteria count amount reaches 55% or more compared with before freeze-drying；Rihizobium japonicum
Freeze dried powder is after 28 DEG C of storage 90d, and living bacteria count amount reaches 20% or more compared with before freeze-drying.With existing root nodule
Bacterium compares, and what living bacteria count reduced after 0122 bacterium of Bw freeze-drying of the present invention lacks.Slow-growing Soybean rhizobia of the present invention
(Bradyrhizobium japonicum) 0122 bacterium of Bw is low temperature resistant, and activity is high after freeze-drying.
0122 resistance to low temperature of present invention Slow-growing Soybean rhizobia Bw is good, is conducive to low temperature process and storage, is suitble to
Be prepared into freeze-dried powder dosage form carry out using.
Detailed description of the invention
Fig. 1 is the colonial morphology figure of Slow-growing Soybean rhizobia (Bradyrhizobium japonicum) Bw 0122.
Specific embodiment
The technical solution of the present invention is not limited to the following list, further includes between each specific embodiment
Any combination.
Specific embodiment 1: present embodiment is suitble to that the rihizobium japonicum of freeze dried powder is made, for slow raw type soybean
Rhizobium (Bradyrhizobium japonicum) Bw 0122, it is general to be preserved in China Committee for Culture Collection of Microorganisms
Logical microorganism center, deposit number are CGMCC No.16010.
The condition of culture of present embodiment Slow-growing Soybean rhizobia Bw 0122 be pH be 6.8~7.0, temperature be 28~
30 DEG C, incubation time be 3~5 days, 0122 culture medium of Slow-growing Soybean rhizobia Bw is according to parts by weight than by 10 parts of sweet dews
Alcohol, 0.4 part of yeast extract extract, 0.1 part of sodium chloride, 0.5 part of dipotassium hydrogen phosphate, 0.2 part of magnesium sulfate, 18 parts of agar and 1000
Part distilled water composition.
Present embodiment Slow-growing Soybean rhizobia separation and Extraction is chosen fresh in the soybean of Qiqihaer City's soybean in field
Full root nodule, is cut with scissors, and first root nodule is placed in sterile water and is impregnated, and removes the impurity on surface, anhydrous with 75%
After ethyl alcohol surface sterilization 3min, then the NaClO for being 3.5% with concentration sterilizes 8min, is rinsed 10 times with sterile water later,
After being cut single root nodule with sterilized knife, then root nodule is clamped on Congo red culture medium with tweezers and is crossed, in 28 DEG C of perseverances
It just has clearly bacterium colony after cultivating 2~3d in warm incubator and grows, and according to the form of thallus, size, transparency, glutinous
The features such as consistency, color, gloss, the scribing line for carrying out 3~4 times to isolated rhizobium monoclonal separates, clear until growing
Then clear monoclonal carries out Blast with the existing sequence in GenBank by 16S rDNA gene sequencing result and compares, should
Sequence and lactobacillus plantarum belong to the homology highest of 16S rDNA gene order, similitude 99%, by combining thalli morphology
Feature, growth conditions, Physiology and biochemistry qualification result determine the bacterial strain referring to describing in primary Jie Shi Bacteria Identification handbook the 8th edition
Belong to rihizobium japonicum, is named as Slow-growing Soybean rhizobia (Bradyrhizobium japonicum) Bw 0122.
Present embodiment Slow-growing Soybean rhizobia (Bradyrhizobium japonicum) Bw 0122 is inoculated in above-mentioned
0122 culture medium of Slow-growing Soybean rhizobia Bw carries out passage assays, and every 2d passage is primary, and continuous passage 10 times, every passage one
Secondary inclined-plane bacterial strain carries out shake flask fermentation test, and bacterial strain Bw 0122 has preferable genetic stability as the result is shown.
Present embodiment Slow-growing Soybean rhizobia (Bradyrhizobium japonicum) Bw 0122 is gram-negative
Property aerobasilus, 0.5~0.9 1.2~6.0 μm of μ m.
Present embodiment Slow-growing Soybean rhizobia (Bradyrhizobium japonicum) 0122 bacterium colony circle of Bw is convex
It rises, white, opaque, surface is smooth, and edge is irregular, as shown in Figure 1.
Specific embodiment 2: application of the rihizobium japonicum as freeze dried powder as described in specific embodiment one.
Specific embodiment 3: the difference of present embodiment and specific embodiment two is: by giving birth to type soybean nodulation slowly
Bacterium (Bradyrhizobium japonicum) Bw 0122 is prepared into freeze dried powder by frozen dried.Other and specific implementation
Mode two is identical.
Present embodiment Slow-growing Soybean rhizobia (Bradyrhizobium japonicum) Bw 0122 is by freeze-drying
Rihizobium japonicum freeze dried powder is made in reason, and wherein rihizobium japonicum (Bradyrhizobium japonicum) Bw 0122 is lyophilized
Method are as follows: fermented using ultracentrifuge to Slow-growing Soybean rhizobia (Bradyrhizobium japonicum) Bw 0122
5~10min of liquid 10000r/min continuous centrifugal goes supernatant to collect thallus, adds according to the ratio of weight fraction ratio 1:1 into thallus
Enter cryoprotector, bacteria suspension is made after concussion uniformly, bacteria suspension is dispensed into 10ml ampulla, and every bottle of 3~5ml is placed in 4 DEG C
It after 30min is pre-chilled in refrigerator, is transferred in freeze dryer again after being transferred to -20 DEG C of pre-coolings, in -8 DEG C of freeze-drying 14h, detects moisture <
2.5% terminates to get rihizobium japonicum freeze dried powder is arrived, and rubber plug is stored spare after sealing with aluminium lid.Wherein bacteria protectant is
Skimmed milk.
Above-mentioned 0122 fermentation liquid of Slow-growing Soybean rhizobia Bw the preparation method comprises the following steps: Slow-growing Soybean rhizobia
(Bradyrhizobium japonicum) Bw 0122 is inoculated into 0122 culture medium of Slow-growing Soybean rhizobia Bw, in pH
It is cultivated 3~5 days under the conditions of being 28~30 DEG C for 6.8~7.0, temperature, wherein 0122 culture medium of Slow-growing Soybean rhizobia Bw is pressed
According to weight fraction ratio by 10 portions of mannitol, 0.4 part of yeast extract extract, 0.1 part of sodium chloride, 0.5 part of dipotassium hydrogen phosphate, 0.2 part
Magnesium sulfate, 18 parts of agar and 1000 parts of distilled water compositions.
By living bacteria count after freeze-drying and before being lyophilized, living bacteria count is compared, and living bacteria count amount remains to reach after freeze-drying
To 92% or more；Rihizobium japonicum freeze dried powder is after 10 DEG C of storage 90d, and living bacteria count amount reaches compared with before freeze-drying
55% or more；Rihizobium japonicum freeze dried powder is after 28 DEG C of storage 90d, and living bacteria count amount reaches compared with before freeze-drying
20% or more.It compares with existing rhizobium, what living bacteria count reduced after the freeze-drying of 0122 bacterium of present embodiment Bw lacks, this
It is low temperature resistant to invent 0122 bacterium of Bw, activity is high after freeze-drying.
Embodiment 1
Utilize rihizobium japonicum (Slow-growing Soybean rhizobia (Bradyrhizobium of the invention within 2018
Japonicum) Bw 0122) freeze dried powder that is prepared tested, specific as follows:
1, place is tested
Qiqihaer City, Heilongjiang Province, scientific base, the base area Fu Laer.123 ° 40 ' of east longitude, north latitude: 47 ° 15 ', height above sea level:
148.3m topography is flat.
2, reagent agent and kind
Medicament: the rihizobium japonicum freeze-dried powder that Slow-growing Soybean rhizobia Bw 0122 of the present invention is obtained by frozen dried
Agent (rihizobium japonicum freeze dried powder is stored at room temperature, 1~5 day holding time).
Soybean varieties: Qi Nong 10.
3, experiment process and design
Plantation plot is positive a batch Soybean Field (first crop crop is corn), and varieties of plant is neat agriculture 10, and test uses great Qu
Comparison, 2 processing do not set repetition, and respectively rihizobium japonicum freeze dried powder is dressed seed, CK (not with kind), each processing area 8
Row × 0.65m × 170m, plot area 884m2It was sowed in the moon 21, emergence on June 6.
4, investigation and pilot project
Obtained by field investigation and indoor species test, analysis: rihizobium japonicum freeze dried powder can make soybean yield-increasing, per hectare
Increase production 184.62kg, rate of growth 5.97% than control, soybean is calculated by 3.7 yuan/kg, and per hectare increases by 683 yuan of benefit, in detail
It is shown in Table 1.
1 field investigation of table and indoor species test table
It can be seen that Slow-growing Soybean rhizobia (Bradyrhizobium japonicum) Bw 0122 of the invention is resistance to
Low temperature performance well, activity is high after freeze dried powder is made, with Slow-growing Soybean rhizobia (Bradyrhizobium of the invention
Japonicum) freeze dried powder made of Bw 0122 carries out crops seed dressing, can effectively raise the yield of crops.
Embodiment 2
Slow-growing Soybean rhizobia (Bradyrhizobium japonicum) Bw 0122 of the invention and existing soybean
Freeze dried powder is made after freeze-drying respectively in rhizobium USDA110 bacterial strain, is then respectively placed in 10 DEG C and 28 DEG C progress
Heat preservation storage test measures its living bacteria count after storing 0d, 30d, 50d, 90d, as a result as shown in table 2 and table 3.Its
In, measuring 0122 bacterium of Bw living bacteria count before freeze-drying is 5.6 × 109Cfu/g, after freeze-drying living bacteria count be 5.4 ×
109Cfu/g, living bacteria count remains to reach 96% after freeze-drying；And USDA110 bacterial strain be lyophilized before living bacteria count be 6.6 ×
109Cfu/g, activity is 5.2 × 10 after freeze-drying9Cfu/g, living bacteria count is only 78.38% after freeze-drying；0122 bacterium of Bw of the present invention
Activity is high after freeze-drying.
2 two kinds of microbial inoculums of table change in 28 DEG C of storage bacterium numbers
| It saves in number of days (day) | 0 | 30 | 50 | 90 |
| Bw0122 | 5.6×109cfu/g | 4.3×109cfu/g | 2.8×109cfu/g | 1.2×109cfu/g |
| USDA110 | 6.6×109cfu/g | 3.4×109cfu/g | 1.8×109cfu/g | 7.2×108cfu/g |
3 two kinds of microbial inoculums of table change in 10 DEG C of storage bacterium numbers
| It saves in number of days (day) | 0 | 30 | 50 | 90 |
| Bw0122 | 5.6×109cfu/g | 4.9×109cfu/g | 4.2×109cfu/g | 3.3×109cfu/g |
| USDA110 | 6.6×109cfu/g | 4.1×109cfu/g | 2.4×109cfu/g | 9.0×108cfu/g |
From table 2 and table 3 as can be seen that Slow-growing Soybean rhizobia (Bradyrhizobium japonicum) Bw of the present invention
The freeze-dried powder preparation of 0122 preparation all has excellent resistance to storage compared with existing rihizobium japonicum under different storage temperatures
Ability and higher activity are deposited, Slow-growing Soybean rhizobia (Bradyrhizobium japonicum) Bw 0122 of the present invention is resistance to
Low temperature performance well.
Claims (2)
1. a kind of rihizobium japonicum for being suitble to that freeze dried powder is made is Slow-growing Soybean rhizobia (Bradyrhizobium
Japonicum) 0122 Bw, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is
CGMCC No.16010。
2. application of the rihizobium japonicum as described in claim 1 as freeze dried powder.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910168059.4A CN109880759B (en) | 2019-03-06 | 2019-03-06 | Rhizobium japonicum suitable for being prepared into freeze-dried powder and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910168059.4A CN109880759B (en) | 2019-03-06 | 2019-03-06 | Rhizobium japonicum suitable for being prepared into freeze-dried powder and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109880759A true CN109880759A (en) | 2019-06-14 |
| CN109880759B CN109880759B (en) | 2022-05-06 |
Family
ID=66930905
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910168059.4A Active CN109880759B (en) | 2019-03-06 | 2019-03-06 | Rhizobium japonicum suitable for being prepared into freeze-dried powder and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109880759B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111960883A (en) * | 2020-07-28 | 2020-11-20 | 沧州市农林科学院 | Special bacterial fertilizer for soybean varieties and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013090884A1 (en) * | 2011-12-16 | 2013-06-20 | Novozymes Biologicals Holdings A/S | Bradyrhizobium strains |
| CN106754500A (en) * | 2016-12-14 | 2017-05-31 | 黑龙江省科学院大庆分院 | The preparation method and applications of alfalfa nodule bacteria research immobilization embedded particles formulation |
-
2019
- 2019-03-06 CN CN201910168059.4A patent/CN109880759B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013090884A1 (en) * | 2011-12-16 | 2013-06-20 | Novozymes Biologicals Holdings A/S | Bradyrhizobium strains |
| CN106754500A (en) * | 2016-12-14 | 2017-05-31 | 黑龙江省科学院大庆分院 | The preparation method and applications of alfalfa nodule bacteria research immobilization embedded particles formulation |
Non-Patent Citations (2)
| Title |
|---|
| ROSALIND DEAKER等人: "Legume seed inoculation technology_a review", 《SOIL BIOLOGY AND BIOCHEMISTRY》 * |
| 朱虹等人: "不同保藏年代的大豆根瘤菌冻干菌种的生物学活性研究", 《土壤肥料》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111960883A (en) * | 2020-07-28 | 2020-11-20 | 沧州市农林科学院 | Special bacterial fertilizer for soybean varieties and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109880759B (en) | 2022-05-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103627662B (en) | A kind of Bradyrhizobium sp Arachis and uses thereof | |
| CN110066756B (en) | Paenibacillus kribbensis and preparation and application thereof | |
| CN112795496B (en) | Paenibacillus polymyxa and application thereof in preventing and treating stem basal rot of Chinese cabbage | |
| CN114875016B (en) | Formulated carrier suitable for pseudomonas fluorescens and microbial inoculum thereof | |
| CN106701631A (en) | Strain of streptomyces roseoflavus and application thereof | |
| CN109749953B (en) | Bacillus cereus, microbial inoculum and preparation method and application thereof | |
| CN110438017A (en) | Branch spore Saksenaea vasiformis bacterial strain LJ1 and its application | |
| CN102586143B (en) | Bacillus mojavensis and microorganism bacterium agent thereof for controlling cucumber downy mildew | |
| CN111869682B (en) | Application of chaetomium globosum in preventing and treating wheat take-all | |
| CN110317747A (en) | A kind of bacillus amyloliquefaciens JT68 and its application in prevention and treatment tea anthracnose | |
| CN109182194A (en) | One plant of Yang Ling rhizobium for promoting coronule flower growth and its cultural method and application | |
| CN110452859B (en) | Bacillus amyloliquefaciens B4216 and application thereof | |
| CN102018000B (en) | Application of BZ6-1 bacterial strain in preparing drugs for treating plant peanut bacterial wilt | |
| CN109880759A (en) | A kind of rihizobium japonicum for being suitble to that freeze dried powder is made and its application | |
| CN115197853B (en) | Endophyte Epicoccum thailandicum LF-28 strain and application thereof | |
| CN108841761B (en) | Method for promoting growth of clover and/or increasing yield of clover and microbial inoculum used by method | |
| CN109266570B (en) | Microbial preparation for antagonizing growth of aspergillus flavus and application | |
| CN103289931B (en) | Bacillus vallismortis strain SJ and application thereof in preparation of tobacco antiviral preparation and promoter | |
| CN116004468A (en) | Salt-tolerant bacillus B13 and application thereof | |
| CN109207392A (en) | One plant of bacillus megaterium and its application in prevention and treatment American ginseng root maize ear rot | |
| CN109207393A (en) | One bacillus amyloliquefaciens and its application in prevention and treatment celery root rot | |
| CN108570426B (en) | Bacterial strain with phosphate solubilizing function, preparation method of microbial inoculum and microbial inoculum | |
| CN114634889B (en) | Bacillus licheniformis nmxc2-1, fermentation product, microbial inoculum and application thereof | |
| CN116426445B (en) | Pseudomonas bacteria NJAU-T102 and application thereof | |
| CN115261249B (en) | Rhizobium pisum and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |