CN105062935B - A kind of rhizobium and its application - Google Patents
A kind of rhizobium and its application Download PDFInfo
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- CN105062935B CN105062935B CN201510585141.9A CN201510585141A CN105062935B CN 105062935 B CN105062935 B CN 105062935B CN 201510585141 A CN201510585141 A CN 201510585141A CN 105062935 B CN105062935 B CN 105062935B
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Abstract
The present invention relates to the technology in improvement salt-soda soil, specifically a kind of rhizobium and its application.Rhizobium (Ensifer sp.) YIC4027, China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC is preserved on June 19th, 2015, address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number is CGMCC No.CGMCC 10994.Strain growth speed of the present invention is fast, saline-alkali tolerant, can remarkably promote the growth of sesbania under greenhouse and crop field environment with the effective symbiotic nitrogen fixation of sesbania, significantly improve the yield of plant above ground, under ground portion, improve the yield and quality of sesbania；Be conducive to plant sesbania with improveing saline and alkaline and other poor soils, reduce fertilizer application, reduce environmental pollution caused by chemical fertilizer, be finally reached and improve saline and alkaline and lean soil, the purpose for promoting ecological recovery and preserving the ecological environment.
Description
Technical field
The present invention relates to the technology in improvement salt-soda soil, specifically a kind of rhizobium and its application.
Background technique
Rhizobium are a kind of gramnegative bacteriums that root nodule or stem tumor row symbiotic nitrogen fixation can be formed with leguminous plant.Work as root
When tumor bacterium and the legume symbiosis to match, effective symbiotic tissues-root nodule or stem tumor are formed, the root in root nodule or stem tumor
The nitrogen in air is converted ammonia by tumor bacterium, is supplied to host plant as nitrogen source, and leguminous plant then supplies rhizobium carbon source
And nutriment, the two form good mutualism relationship.
Since rhizobium and leguminous plant need to undergo a series of interactive molecule dialogues in Interaction, and it is this total
Raw process will receive the influence of environmental factor again, thus original inhabitants high-efficiency nitrogen-fixing rhizobium will preferably adapt to local environment, and work as
Ground leguminous plant forms efficient Symbiotic association.
Sesbania is a kind of salt tolerant, drought resisting, leguminous forage waterlogging, impoverishment tolerant is very capable, it is in alkaline land improving
Play very important effect.Its cauline leaf is not only good green manure, moreover it is possible to make the feed of cattle and sheep, the stalk of stem's lignifying is
The good material of papermaking, seed can squeeze sesbania gum, be used for oil extraction in oil field.
Sesbania is usually used in the improvement in salt-soda soil, and not only salt alkali content is high in salt-soda soil, but also faces and lack the source of manure.And root nodule
Bacterium can provide nitrogen source for sesbania, sesbania is promoted to grow up healthy and sound, promote soil fertility with sesbania symbiosis, in plantation sesbania improvement
Its important function is played in salt-soda soil.
Summary of the invention
The object of the present invention is to provide a kind of rhizobium and its applications in plantation sesbania improvement salt-soda soil.
To achieve the above object, the technical solution adopted by the present invention are as follows:
A kind of rhizobium, rhizobium (Ensifer sp.) YIC4027 have been preserved in the micro- life of China on June 19th, 2015
Object culture presevation administration committee common micro-organisms center (abbreviation CGMCC, address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
No. 3), deposit number is CGMCC No.10994.
Separation screening obtains in the sesbania root nodule that the rhizobium are planted by Huanghe delta salt affected soil, is a plant height
Imitate the strong fast-growing Rhizobium of fixed nitrogen, salt tolerant alkali ability.Housekeeping gene recA sequence is carried out to separating obtained bacterial strain to divide
Analysis, and by GenBank as a result, it has been found that, the recA gene and Ensifer sojae CCBAU of rhizobium of the present invention
05684TIt is 93.8% like property highest.So that it is determined that the rhizobium YIC4027 separated is the potential novel species of Ensifer, and
It is named as Ensifer sp.YIC4027.
The application of rhizobium, application of the rhizobium in improvement salt-soda soil.
Further, application of the rhizobium in the salt-soda soil of improvement plantation sesbania.
It is further that the rhizobium are incubated at gained bacterium solution in fluid nutrient medium culture and are sprayed at Sesbania seed
The application in the salt-soda soil of the Huanghe delta is then planted on surface.
Moreover, the rhizobium are incubated at YMA fluid nutrient medium to logarithmic growth phase, the sprinkling of gained bacterium solution
On Sesbania seed surface, the application in the salt-soda soil of the Huanghe delta is then planted.
The present invention is had an advantage in that
1. rhizobium YIC4027 of the invention has efficient nodulation and nitrogen fixation growth-promoting characteristic, being inoculated with the bacterium can be significantly improved
The 33.86% of sesbania biomass, growth-promoting significant effect can reach volume increase purpose；
2. sesbania root week soil salinity reduces by 20%, and Soil Available nitrogen increases after being inoculated with rhizobium plantation sesbania of the present invention
Add 11.9%, rapid available phosphorus increases by 17.72, and available potassium increases by 18.52%, can effectively improve salt-soda soil；
3. rhizobium YIC4027 of the invention, which is suitable for plantation sesbania, improves Huanghe delta salinized soil, have very big
Application and popularization value.
Detailed description of the invention
Fig. 1 is the bacterium colony photo of bacterial strain YIC4027 provided in an embodiment of the present invention.
Fig. 2 promotes field in the field plot trial for inoculating strain YIC4027 kind provided in an embodiment of the present invention plant sesbania
Cyanines above and below ground part biological amount increases effect.
Fig. 3 is that inoculating strain YIC4027 kind provided in an embodiment of the present invention plants promotion sesbania stem in the plot experiment of sesbania
Rugosity increases effect.
Fig. 4 is that inoculating strain YIC4027 kind provided in an embodiment of the present invention is planted in the plot experiment of sesbania to sesbania main root
The increase effect of lateral root growth.
Specific embodiment
Below with reference to embodiment, the invention will be further described.Embodiment is intended to carry out citing description to the present invention, and
It is non-to limit the invention in any form.
Embodiment 1: the acquisition of rhizobium
Ground, the eugonic sesbania of underground part are chosen from Huanghe delta salinized soil planting site, acquires root nodule, it is right
After root nodule carries out surface sterilization, root nodule to be smashed to pieces, and leachate is coated on YMA plate, 28 DEG C of inversions are cultivated 3-5 days, to
When growing the circular colonies that neat in edge, milky are translucent, exocellular polysaccharide is more, picking single colonie and repeatedly scribing line are until pure
Change (referring to Fig. 1).
YMA solid medium (yeast extract mannite agar culture medium) formula are as follows: mannitol 10g/L, yeast extract
Object 3g/L, K2HPO40.25g/L, KH2PO40.25g/L, MgSO40.2g/L, CaCO33g/L, NaCl 0.1g/L, deionization
Water constant volume stops 1000mL, and dispenses respectively into triangular flask, the agar powder of every bottle of final concentration of 20g/L of addition.
1) identification of rhizobium
Gram's staining and Morphological Identification: it is feminine gender that above-mentioned purifying, which is obtained bacterial strain through Gram's staining result, is not generated
Gemma can move, and bacterium colony is wet, median rise, neat in edge；Exocellular polysaccharide content is higher, is typical rhizobium feature.
Molecular Identification: the phyletic evolution status in order to determine this implementation rhizobium strains YIC4027, to its housekeeping gene
The sequence of recA carries out sequencing analysis.
The total DNA of the bacterial strain is extracted first, then uses the universal primer recA41F/recA640R of housekeeping gene recA
PCR amplification is carried out, and PCR product is analyzed.Sequence is as shown in the sequence 1 of list, with Ensifer sojae
CCBAU05684TSimilitude highest similitude is 93.8%, so that it is determined that bacterial strain YIC4027 is the potential novel species of Ensifer.
The recA sequence of Ensifer sp.YIC4027:
In summary qualification result names bacterial strain YIC4027 for Ensifer sp；Ensifer sp.YIC4027,
China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, ground are preserved on June 24th, 2015
Location are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number is CGMCC No.10994.Ensifer sp.YIC4027,
Abbreviation bacterial strain YIC4027.By the above-mentioned screening and identification according to chamber planting sesbania Rhizobium Inoculation, by efficient nodulation and nitrogen fixation bacterial strain
It is named as YIC4027.
Above-mentioned rhizobium strains YIC4027 and sesbania symbiotic nodulation and nitrogen fixation ability is strong, does not carry out genetic modification.Work as thallus
It is harmless to people, animals and plants after being released into natural environment, it is free from environmental pollution, and the growth of sesbania can be remarkably promoted.
The laboratory preservation mode of gained Ensifer sp.YIC4027 rhizobium strains:
Short term storage mode is to be seeded to YMA solid slope, is preserved in 4 DEG C of refrigerators after bacterial strain is grown well, this preservation side
The formula no more than 3 moon；
Long term storage mode are as follows: rhizobium thallus is added in final concentration of 20% glycerol and mixes simultaneously preservation to -80 DEG C of ice
In case, this preservation mode about can be preservation 10 years.
2) the saline-alkali tolerant capability analysis of bacterial strain YIC4027
Bacterial strain YIC4027 is cultivated into the culture medium of different salt concentration gradients and measures the salt tolerant of the bacterium and can be carried out experiment
Analysis, the results showed that the bacterium is resistant to 5% and salinity below；It is well-grown in 6-10 culture medium in pH.Therefore should
Bacterium has stronger salt tolerant alkali ability.
The microbial inoculum application and preparation of 2 bacterial strain YIC4027 of embodiment
1) bacterial strain activates
With the oese picking YIC4027 bacterium colony after sterilizing, and cross on YMA solid medium, 28 DEG C of inversions cultures
2-3 days until bacterium colony is grown.
2) expand culture
With one to the two ring thallus of oese picking after sterilizing, it is seeded to the liquid YMA fluid nutrient medium after 200mL sterilizes
In, 28 DEG C, 160 revs/min shaken cultivation 24~48 hours, culture is to OD600 >=1.5, as stand-by microbial inoculum.By microbial inoculum put to
In 4 DEG C of refrigerators, it can be reserved for 1 week.
3) application of rhizobium
Experimental period is mid or late May to early October.It levels land, and ridging, ridge spacing about 1.6m.
Healthy, full, even particle size the Sesbania seed of picking, is placed in clean plastic tub.
Above-mentioned acquisition microbial inoculum is sprayed on Sesbania seed surface with small sprayer at shady and cool without sunlight, and with spades or its
He stirs evenly at tool, but protects and is all stained with enough microbial inoculums on every seed.
The seed for being stained with nitragin will be dried in the air to adhesion in the cool preferably in favor of sowing, should not be to sudden and violent under sunlight
It shines, otherwise rhizobium can be killed by the ultraviolet light in sunlight.
4. sowing
Experimental group uniformly sows the seed for being sprayed with nitragin into the experimental plot put in order, line space 30cm,
Spacing in the rows 10cm, depth of planting 5cm.
Experimental comparison group does not spray nitragin, directly sows seed into the experimental plot put in order, line-spacing, spacing in the rows,
Planting depth is all identical as experimental group.
After finishing, broad irrigation is used immediately, the reason is that sesbania kind skin is thicker, needs flood pouring that can just promote its germination,
If will plant again after seed warm water swelling in advance, ground moistening can be only kept.
5. field management
Sesbania tolerance soil depletion ability is stronger, does not need generally to apply fertilizer.Resistance to pest and disease damage does not need generally to spray insecticide.Seedling
Phase pays attention to weeding out the rank grass, and sesbania drought resisting, waterlogging ability are stronger, generally no longer needs to water when the Huanghe delta is planted, in the summer
There are apparent rainy season in season, the Huanghe delta, and sesbania can be good at the influence for being resistant to rainfall.Plot experiment bacterial strain as the result is shown
It is 100% that YIC4027 accounts for ratio of outflow in the experimental plot of the Huanghe delta, there is very strong marker technique in situ.Plantation sesbania is being received
Obtaining early October in season is sampled analysis to sesbania and root week 5cm or so soil respectively (referring to table 1 and Fig. 2,3,4).
1 soil physico-chemical property of table
To dry weight, the main side of sesbania aerial part plant height, stem thickness, aerial part dry weight, chlorophyll content and under ground portion
Root long degree is for statistical analysis, test result such as Fig. 1,2,3 and table 1 shown in, compared to the sesbania of not Rhizobium Inoculation, be inoculated with root nodule
The Aboveground Biomass of Young dry weight of sesbania increases by 33.86%, stem thickness degree and increases by 36.79% after bacterium；Furthermore inoculating strain YIC4027
The root system of sesbania is more flourishing afterwards, and root weights increase by 72.73%, main root length and increase by 17.98%, and lateral root length increases
10.06%, as shown in table 1, compared to the sesbania of not Rhizobium Inoculation, or so sesbania root week 5cm soil after inoculating strain YIC4027
Nutrient dramatically increases, and available nitrogen increases by 11.9%, rapid available phosphorus and increases by 17.72%, available potassium increase by 18.52%, soil salt
Concentration reduces by 20%.Therefore, bacterial strain YIC4027 can be remarkably promoted in Huanghe delta plantation sesbania improvement salt-soda soil application
The growth of sesbania and the raising of biomass, to significantly play the purpose of volume increase；In addition, the salinity in salt-soda soil significantly reduces,
Soil fertility is obviously improved.Thus inoculation YIC4027 can make during Huanghe delta plantation sesbania improvement salinized soil
For one of Normal culture technique.
Claims (1)
1. a kind of application of rhizobium, it is characterised in that: rhizobium are incubated at YMA fluid nutrient medium to logarithmic growth phase, institute
It obtains bacterium solution and is sprayed at Sesbania seed surface, then plant the application in the salt-soda soil of the Huanghe delta；
The rhizobium (EnsiferSp.) YIC4027 is preserved in Chinese microorganism strain preservation on June 19th, 2015
Administration committee's common micro-organisms center (abbreviation CGMCC, address are as follows: the institute 3 of BeiChen West Road, Chaoyang District, BeiJing City 1),
Deposit number is CGMCC No.10994.
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| CN106358661B (en) * | 2016-08-27 | 2020-04-17 | 山东省潍坊市农业科学院 | Method for cultivating rhizobium mother strain |
| CN107056521A (en) * | 2016-10-19 | 2017-08-18 | 宁波创蓝环境科技有限公司 | A kind of microbial manure and its manufacture method that getting fat is improved for salt affected soil |
| CN112779177A (en) * | 2019-11-07 | 2021-05-11 | 中国科学院烟台海岸带研究所 | Compound microbial agent special for saline-alkali soil and application thereof |
| CN113088510B (en) * | 2021-04-14 | 2022-11-18 | 上海健康医学院 | Agrobacterium tumefaciens, immobilized bacterium thereof and method for degrading azithromycin |
| CN113179882A (en) * | 2021-04-29 | 2021-07-30 | 长江师范学院 | Method for promoting nodulation and nitrogen fixation of root system of tumorous stem mustard by utilizing sesbania azorhizobium |
| CN114045240B (en) * | 2021-11-18 | 2023-07-14 | 中国林业科学研究院森林生态环境与保护研究所 | Rhizobium, microbial inoculum prepared from rhizobium and application of rhizobium |
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