CN107164261A - Rhizobium and its application that one plant of promotion villose vetch increases - Google Patents
Rhizobium and its application that one plant of promotion villose vetch increases Download PDFInfo
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- CN107164261A CN107164261A CN201710354501.3A CN201710354501A CN107164261A CN 107164261 A CN107164261 A CN 107164261A CN 201710354501 A CN201710354501 A CN 201710354501A CN 107164261 A CN107164261 A CN 107164261A
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- rhizobium leguminosarum
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/41—Rhizobium
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention discloses one plant of rhizobium leguminosarum for promoting villose vetch to increase and its application.The bacterial strain number of the rhizobium leguminosarum is m1 10 3, and it is CGMCC No.11877 in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center.It is demonstrated experimentally that plant height, plant fresh weight are compared and dramatically increased with rhizobium leguminosarum ACCC16505 plant height, plant fresh weight than not connecing bacterium control, inoculation rhizobium leguminosarum H10 after villose vetch inoculation rhizobium leguminosarum m1 10 3.The present invention has broad application prospects in villose vetch planting industry.
Description
Technical field
The present invention relates to one plant in the field of agricultural microorganism rhizobium for promoting villose vetch to increase and its application.
Background technology
Villose vetch (Vicia villosa Roth, the also referred to as pale reddish brown sweet potato of hair leaf, abbreviation hair sweet potato) is a kind of the green of high-quality
Fertile crop, is mainly distributed on China the Yellow River, Huaihe River, the band of Haihe basin one, and also there was sowing on the ground such as distant year, Inner Mongol, Xinjiang in recent years,
Planted area is larger.The cold tolerance of villose vetch is stronger, and general kind is resistant to subzero 20 DEG C of low temperature of short time, and seedling
Wintering rate it is very high, while villose vetch is drought-enduring and impoverishment tolerant is also very strong, typically in barrenr grown on soil, can also receive
To higher yield, adaptability is wider.
For culture fertility, it is ensured that Sustainable Agricultural stable development, the Ministry of Agriculture is determined since summer nineteen ninety-five in whole nation implementation
To manufacture " fertile_soil plan " of the organic fertilizer as main contents, be green manure crop be again legume villose vetch cultivated area
It is being continuously increased.The research and application of Rhizobium leguminosarum worldwide have more than 100 years history, almost in whole world model
All advocated in enclosing and legume inoculation is carried out to legume.
Nitrogen is one of most important nutrient in plant growth, and rhizobium are that a class can infect legume root
(minority is stem) forms the bacterium that root nodule carries out biological nitrogen fixation, and rhizobium and legume symbiosis system are works in biological nitrogen fixation
With most strong system, the nitrogen in air is converted into the ammonia of plant available, the nitrogen fixed by the nitrogen-fixing microorganism in soil
About the 65% of biological nitrogen fixation total amount.Currently, legume is planted in degenerated soil increasingly to be paid close attention to by people, because
Nitrogen nutrition is poorer in degenerated soil, and soil fertilizer can be improved by the symbiotic azotification of legume and rhizobium
Power, Rhizobium Inoculation can improve yield and the nitrogen reserves of legume.If the legume of plantation is without corresponding high
Nitrogen of the Rhizobium strains therewith in symbiosis dross and fixed air is imitated, they are by the reference state nitrogen being completely dependent in soil, not only
Can not supplement can consume the nitrogen in soil on the contrary, cause soil fertility to decline.Therefore, in order to give full play to rhizobium and pulse family
The symbiotic azotification of plant, to maintain soil nitrogen balance, it is necessary to which screening high efficient strain carries out artificial infection.Development and utilization
Legume and the potentiality of rhizobium Symbiotic nitrogen-fixing effect are very big.During biological nitrogen fixation, according to rhizobium to host plant
Selectivity, the plant suitable for same rhizobium nodulation and nitrogen fixation is classified as family, rhizobium can turn in race between each plant
Connect, referred to as cross inoculation group.Villose vetch and pea are above cross inoculation groups in rhizobium application, and rhizobium leguminosarum can both be inoculated with not
Different cultivars pea can also be inoculated with villose vetch, but effect of inoculation differs greatly.
There is data to suggest that between different green manure species, the nitrogen fixing capacity of villose vetch is highest.While villose vetch
Soil building is acted on and green manuring, the yield increasing effect of stub land are all very significant.Nearly ten years, China villose vetch cultivated area
Constantly expand, the cultivation of villose vetch and gradually stepped up using technology, the research and application of villose vetch Rhizobium Inoculation technology
It is enlarged attention.
The content of the invention
The technical problems to be solved by the invention are how to remarkably promote villose vetch growth.
In order to solve the above technical problems, the invention provides one plant of rhizobium.
Rhizobium provided by the present invention are rhizobium leguminosarum (Rhizobium leguminosarum), and its bacterial strain number is
M1-10-3, the bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on December 14th, 2015
(abbreviation CGMCC, address is at center：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number is CGMCC No.11877,
Hereinafter referred to as rhizobium leguminosarum m1-10-3.
Rhizobium leguminosarum m1-10-3 belong to Gram-negative, it is short and small shaft-like, thalline dyeing it is uneven, formed coloring with
The ring bodies of not colored part, non-staining part lipid content is higher.Without gemma, tool end life flagellum or peritrichous can be transported
It is dynamic.Thalline size is (0.5-0.8) × (1.1-2.9) microns.Grown on yeast juice mannite agar culture base plane, bacterium colony
Rounded projection, neat in edge is smooth compared with wetted surface, and quality is homogeneous, and light milky white, lawn is sticky.Rhizobium leguminosarum m1-
10-3 has the 16S rDNA sequences of sequence 1 in sequence table.
In order to solve the above technical problems, present invention also offers the microbial inoculum for promoting villose vetch growth.
The microbial inoculum provided by the present invention for promoting villose vetch to grow, contains rhizobium leguminosarum m1-10-3 or/and pea root
Knurl bacterium m1-10-3 metabolin.
The microbial inoculum for promoting villose vetch growth concretely improves villose vetch individual plant weight and/or improves hair leaf sweet potato
The microbial inoculum of sub- plant height.The villose vetch individual plant weight refer to villose vetch individual plant aerial part and under ground portion weight it
With.
The active component of above-mentioned microbial inoculum can be rhizobium leguminosarum m1-10-3 or/and rhizobium leguminosarum m1-10-3 metabolism
Thing, the active component of above-mentioned microbial inoculum can also contain other biological composition or abiotic component, the other active components of above-mentioned microbial inoculum
Those skilled in the art can determine according to microbial inoculum to villose vetch Plant weight and/or plant height facilitation effect.The microbial inoculum
It may also include carrier.The carrier can be solid carrier or liquid-carrier.The solid carrier is mineral material, biomaterial；
The mineral material can be at least one in turf, clay, talcum, kaolin, montmorillonite, white carbon, zeolite, silica and diatomite
Kind；The biomaterial is the stalk of all kinds of crops, loose shell, straw, peanut shell, corn flour, bean powder, starch, turf and animal
At least one of excrement；The liquid-carrier can be water；In the microbial inoculum, rhizobium leguminosarum m1-10-3 or/and pea root
Knurl bacterium m1-10-3 metabolin can be with the living cells being cultured, the zymotic fluid of living cells, the filtrate of cell culture or cell
Exist with the form of the mixture of filtrate.The formulation of the microbial inoculum can be a variety of formulations, such as liquor, emulsion, suspending agent, pulvis,
Granule, wettable powder or water dispersible granules.
Wherein, rhizobium leguminosarum m1-10-3 metabolin is that rhizobium leguminosarum m1-10-3 ferments in microbial liquid to train
Support in base and cultivate obtained material.
As needed, surfactant (such as polysorbas20, Tween 80), adhesive, stably can be also added in the microbial inoculum
Agent (such as antioxidant), pH adjusting agent.
The microbial inoculum of rhizobium leguminosarum m1-10-3 or described promotions villose vetch growth is preparing promotion villose vetch growth
Application in product and the application in villose vetch growth is promoted belong to protection scope of the present invention.
In above-mentioned application, the promotion villose vetch growth can be raising villose vetch individual plant weight and/or raising hair leaf
Sweet potato plant height.
In above-mentioned application, the product for promoting villose vetch growth can be the microbial inoculum for promoting villose vetch to grow or contain
The bio-feritlizer of the microbial inoculum for promoting villose vetch to grow.
In the application, the villose vetch can be Turkmenistan hair sweet potato, Qinghai sweet potato, and/or blue or green sweet potato one.
In order to solve the above technical problems, present invention also offers a kind of method for cultivating rhizobium leguminosarum m1-10-3.
Culture rhizobium leguminosarum m1-10-3 provided by the present invention method, is included in the culture for cultivating rhizobium
The step of rhizobium leguminosarum m1-10-3 being cultivated in base.
The culture medium for being used to cultivate rhizobium can be prepared as follows：Glycerine 3-5ml, mannitol 2-5g,
Yeast extract 0.8-1g, K2HPO40.5-1g, anhydrous MgSO40.1-0.2g, CaSO4·2H2O 0.1-0.2g, NaCl0.1-
0.2g, 1% (NH4)6Mo7O24·4H2O 1-1.5ml, 1%H3BO31-1.5ml, agar 20g, 1000ml is settled to water, is adjusted
PH value is 6.8-7.0, and sterilizing obtains the culture medium for cultivating rhizobium.
It is demonstrated experimentally that the present invention rhizobium leguminosarum m1-10-3 to villose vetch than rhizobium leguminosarum H10 and pea root nodule
Bacterium ACCC16505 has more significant promotion growth and effect of increasing production.Rhizobium leguminosarum m1-10-3 is combined with villose vetch inoculation
There is significant effect of increasing production to villose vetch, with higher practicality and replicability, be expected to develop into villose vetch
A new excellent combination technique in rhizobium application field.The present invention has before wide application in villose vetch planting industry
Scape.
Preservation explanation
Strain name：Rhizobium leguminosarum Rhizobium leguminosarum
Strain number：m1-10-3
Preservation mechanism：China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is referred to as：CGMCC
Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date：On December 14th, 2015
Collection is registered on the books numbering：CGMCC No.11877
Embodiment
The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the scope being not intended to be limiting of the invention.Experimental method in following embodiments, unless otherwise specified, be
Conventional method.Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
Rhizobium leguminosarum (Rhizobium leguminosarum) ACCC 16505 in following embodiments is in nineteen ninety 4
It is concealed within 1st China Committee for Culture Collection of Microorganisms agriculture microorganism center (abbreviation ACCC, address the moons：Beijing sea
Shallow lake area Zhong Guan-cun South Street 12, INST OF AGRICULTURAL RESOURCES, postcode 100081), certainly should
The public can obtain the bacterial strain from the agriculture microorganism center of China Committee for Culture Collection of Microorganisms from collection day.Pea root nodule
Bacterium (Rhizobium leguminosarum) ACCC 16505 hereinafter abbreviation rhizobium leguminosarum ACCC16505.
The compound method of solid medium in following embodiments is as follows：Glycerine 5ml, mannitol 5g, yeast extract 1g,
K2HPO40.5g, anhydrous MgSO40.2g, CaSO4·2H2O 0.2g, NaCl 0.1g, 1% (NH4)6Mo7O24·4H2O1ml、
1%H3BO31ml, agar 20g, 1000ml is settled to water, and tune pH value is 6.8-7.0,121 DEG C of sterilizing 30min.
The compound method of fluid nutrient medium in following embodiments is as follows：Glycerine 5ml, mannitol 5g, yeast extract 1g,
K2HPO40.5g, anhydrous MgSO40.2g, CaSO4·2H2O 0.2g, NaCl 0.1g, 1% (NH4)6Mo7O24·4H2O1ml、
1%H3BO31ml, 1000ml is settled to water, and tune pH value is 6.8-7.0,121 DEG C of sterilizing 30min.
Embodiment 1, rhizobium leguminosarum (Rhizobium leguminosarum) m1-10-3CGMCC11877 separation and
Identification
1st, the separation of bacterial strain
Wild villose vetch root nodule is gathered from Hai Dongshi safeties area of Qinghai Province, (mannitol 10g, yeast extract are taken with flat board
1g, dipotassium hydrogen phosphate 0.5g, calcium sulfate 0.2g, magnesium sulfate 0.2g, sodium chloride 0.1g, mass content are water-soluble for 1% ammonium molybdate
The liquid 1ml and boric acid aqueous solution 1ml that mass content is 1%, 0.5% Congo red 1ml, 20g agar is settled to 1L, pH value with water
For 6.8-7.0) the isolated bacterial strain m1-10-3 of line.
2nd, the identification of bacterial strain
2.1st, Morphological Identification
Exponential phase will be in, and bacterium colony size is stable, above-mentioned steps 1 separate and purified obtained bacterial strain m1-10-3
Carry out single bacterium colony state description, the main size including bacterium colony, color, transparency, wettability, bacterium colony surface state, bacterium colony side
Edge state.On the other hand, to the bacterial strain m1-10-3 in exponential phase, observation by light microscope is used after smear staining
The form of thalline.
As a result show that bacterial strain m1-10-3 belongs to Gram-negative, short and small shaft-like, thalline dyeing is uneven, forms coloring
With the ring bodies of not colored part, non-staining part lipid content is higher.Without gemma, tool end life flagellum or peritrichous, energy
Motion.Thalline size is (0.5-0.8) × (1.1-2.9) microns.Grown on yeast juice mannite agar culture base plane, bacterium
Fall rounded projection, neat in edge is smooth compared with wetted surface, and quality is homogeneous, and light milky white, lawn is sticky.
2.2nd, 16S rDNA sequence homology analysis
Using the bacterial strain m1-10-3 of the gained of colony polymerase chain reaction (PCR) method amplification step 1 16S rDNA fragments, related reagent is by complete
Formula King Company provides.16S rRNA genetic fragments are expanded and the result of cloning and sequencing shows, bacterial strain m1-10-3 16S
RDNA has the nucleotide sequence of sequence 1 in sequence table.Bacterial strain m1-10-3 rRNA genetic fragments and rhizobium leguminosarum
(Rhizobium leguminosarum) bacterial strain CCBAU 85022 16S rRNA Gene Partials sequences (Sequence ID:
EU256422.1 similitude) is up to 99%.
2.3rd, physiological and biochemical property is identified
With reference to《Common bacteria system identification handbook》(east show pearl, the wonderful English common bacterias system identification handbook Beijing of Cai:Section
Learn publishing house, 2011.) and《Microbiology Experiment》(Shen Ping, Fan Xiurong, Li Guang force Microbiology Experiment (third edition) Beijing:
Higher Education Publishing House, 1999.) determine bacterial strain m1-10-3 physiological and biochemical property.As a result show bacterial strain m1-10-3 to change energy
Heterotroph, can by the use of various carbohydrate and organic acid salt as carbon source, if utilizing glucose, lactose, D-ribose, D-
Cellobiose, D-R, mannitol, xylose, D- galactolipins, fructose, dulcitol and inositol；Ammonium salt, nitrate can be utilized
Nitrogen source is used as with most amino acid；The Congo red suction micro- suction color of colour response；Litmus milk reaction is non-condensing, does not produce acid；It can not utilize
Cellulose and starch；It is unable to hydrolyzed casein；3- ketone group lactose is not utilized；Acid is produced from mannitol；Normal containing being grown on sugar culture-medium
With abundant extracellular mucus；Do not produce hydrogen sulfide；Precipitation is not produced in calcium glycerophosphate culture medium.
In view of above-mentioned form, analysis of physio biochemical characteristics and 16s rDNA sequence homology analysis results, step 1 is separated
Purify obtained bacterial strain m1-10-3 and be accredited as rhizobium leguminosarum (Rhizobium leguminosarum).The rhizobium leguminosarum
(Rhizobium leguminosarum) m1-10-3 is preserved in Chinese microorganism strain preservation pipe on December 14th, 2015
(abbreviation CGMCC, address is reason committee common micro-organisms center：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), preservation is compiled
Number be CGMCC No.11877, hereinafter referred to as rhizobium leguminosarum m1-10-3.
The preparation of embodiment 2, rhizobium leguminosarum m1-10-3 microbial inoculums
1st, rhizobium leguminosarum m1-10-3 inclined-plane culture
The rhizobium leguminosarum m1-10-3 of picking embodiment 1 is inoculated in solid medium and carries out inclined-plane culture, is trained at 28 DEG C
Support 56 hours, obtain the rhizobium leguminosarum m1-10-3 of inclined-plane culture.
2nd, the activation of strain
The rhizobium leguminosarum m1-10-3 of picking step 1 inclined-plane culture, is inoculated in 500mL fluid nutrient mediums, 28
Cultivated 48 hours at DEG C, obtain rhizobium leguminosarum m1-10-3 bacterium solutions.
3rd, seed tank culture
The rhizobium leguminosarum m1-10-3 bacterium solutions of 3L steps 2 are taken, are accessed in 100L seeding tanks (containing 60L Liquid Cultures
Base) seed culture is carried out, shaken cultivation is cultivated for 56 hours under 30 DEG C, 120rpm, obtains rhizobium leguminosarum m1-10-3 seeds
Liquid.
4th, fermentation tank culture
The rhizobium leguminosarum m1-10-3 seed liquors of step 3 are accessed in 1000L fermentation tanks in 10% ratio (volume ratio)
(containing 600L fluid nutrient mediums) carries out fermented and cultured, and shaken cultivation 56 hours, obtain rhizobium leguminosarum under 28 DEG C, 150rpm
Rhizobium leguminosarum m1-10-3 content is 3,000,000,000 cfu/mL in m1-10-3 zymotic fluids, the rhizobium leguminosarum m1-10-3 zymotic fluids.
5th, the preparation of rhizobium leguminosarum m1-10-3 microbial inoculums
Crushed after turf is spontaneously dried, cross 100 mesh sieves, then adjusted pH value to be 6.8 with limewash, gone out at 121 DEG C
Bacterium 60min, obtains sterile turf.
The rhizobium leguminosarum m1-10-3 zymotic fluids of step 4 and the sterile turf are mixed, then the propagation training under the conditions of 28 DEG C
48h is supported, rhizobium leguminosarum m1-10-3 microbial inoculums, packing storage is obtained.Finished product is qualified after testing, the rhizobium leguminosarum m1-10-3 bacterium
The content of rhizobium leguminosarum m1-10-3 in agent is 2.0 × 108Cfu/ grams.
Embodiment 3, rhizobium leguminosarum m1-10-3 are inoculated with the water culture experiment of villose vetch
Using filter paper bridge test tube water culture.Filter paper is cut into strip, M types are made, middle recess is according to the size system of seedling
Into V-type aperture, the height of M type filter paper subtracts 4cm for the length of test tube.Test tube used is 2.0cm × 20cm.
The nitrogen-free nutrient solution prepared is respectively charged into the test tube with filter paper supporter, its height is located at H type filter paper
Recess, each processing repeats 4 test tubes, and sets control tube 4, it is filling after sealed with the plastic sheeting of high temperature high voltage resistant after,
15 pounds of 121 DEG C of sterilizing 1h are standby.
Uniform Turkmenistan hair sweet potato seed is selected, 0.1%HgCl is used after soaking 5min with 95% ethanol2Surface sterilizing
5min, then with aseptic water washing 10 times.The seed of sterilization is placed in 28-30 DEG C of incubators and is incubated vernalization.Illumination is required from top
Irradiation, root lucifuge can be placed in greenhouse or lighting box, and intensity about 7000lux -8000lux is shone in illumination；Temperature：25—30
℃；Vernalization length is 0.8-1.5cm.
Experiment, which is set, does not connect bacterium control treatment and rhizobium leguminosarum m1-10-3 processing.Experiment sets three repetitions, repeats every time
Experimental method is as follows：
Rhizobium leguminosarum m1-10-3 processing：10 successful seeds of vernalization are put into bacteria suspension (to be diluted with fluid nutrient medium
The rhizobium leguminosarum m1-10-3 zymotic fluids of the step 4 of embodiment 2 to rhizobium leguminosarum m1-10-3 content are 2.0 × 107cfu/
ML obtains bacteria suspension) middle immersion 30min, seed is carefully pressed from both sides out with sterile tweezers and is put into fixation in the filter paper bridge of water planting liquid test tube
Good sowing, bacteria suspension is averagely sucked in test tube.Sealing is after illumination cultivation in lighting box, and illumination is irradiated from top, during illumination
Between 14h/10h, root lucifuge, intensity of illumination be 7000lux -8000lux；Temperature：25—30℃.
Bacterium control treatment is not connect：Bacteria suspension is replaced with into liquid with differing only in for rhizobium leguminosarum m1-10-3 processing
Culture medium, other operations are identical.
Table 1, Turkmenistan hair sweet potato inoculation rhizobium leguminosarum m1-10-3 water culture experiment results
As a result as shown in table 1, show rhizobium leguminosarum m1-10-3 being inoculated with after the hair sweet potato of Turkmenistan, plant height, plant root
Long, plant fresh weight and plant weights ratio do not connect bacterium control and increase by 32.56%, 18.01%, 19.32% and 47.62%, table respectively
The bright rhizobium leguminosarum m1-10-3 for being inoculated with villose vetch of the invention has significant growth-promoting effect.
Comparative example 1, rhizobium leguminosarum (Rhizobium leguminosarum) H10CGMCC No.11878 separation and
Identification
1st, the separation of bacterial strain
Wild pea root nodule is gathered from Qinghai Province's Ledu County, (mannitol 10g, yeast extract 1g, phosphoric acid hydrogen two are taken with flat board
Potassium 0.5g, calcium sulfate 0.2g, magnesium sulfate 0.2g, sodium chloride 0.1g, ammonium molybdate aqueous solution 1ml and quality of the mass content for 1%
Content is 1% boric acid aqueous solution 1ml, and 0.5% Congo red 1ml, 20g agar is settled to 1L with water, and pH value is 6.8-7.0) draw
The isolated bacterial strain H10 of line.
2nd, the identification of bacterial strain
2.1st, Morphological Identification
Exponential phase will be in, and bacterium colony size is stable, above-mentioned steps 1 separate and purified obtained bacterial strain H10 and carry out
Single bacterium colony state description, the main size including bacterium colony, color, transparency, wettability, bacterium colony surface state, colony edge shape
State.On the other hand, to the bacterial strain H10 in exponential phase, the shape of observation by light microscope thalline is used after smear staining
State.
As a result show that bacterial strain H10 belongs to Gram-negative, small rod-short, thalline size is 0.5~0.9 micron × 1.2
~6.0 microns, the thalli morphology grown in different environments is different.Typically contain Poly-β-hydroxybutyric Acid salt particle.Without gemma,
Have end life flagellum or peritrichous, motion is aerobic.25~30 DEG C of optimum growth temperature, optimal pH 6.0~7.0.Bacterium colony is in circle
Shape, neat in edge, microprotrusion, semi-transparent clear or light milky white, it is sticky.It is flat in yeast juice mannitol inorganic salts agar medium
2~4mm of diameter after being grown 3-6 days on plate.Growth-gen on containing sugar culture-medium is often accompanied by abundant extracellular mucus.
2.2nd, 16S rDNA sequence homology analysis
Using the bacterial strain H10 of the gained of colony polymerase chain reaction (PCR) method amplification step 1 16S rDNA fragments, related reagent is by Quan Shijin
Company provides.16S rRNA genetic fragments are expanded and the result of cloning and sequencing shows, bacterial strain H10 16S rDNA have
The nucleotide sequence of sequence 2 in sequence table.Bacterial strain H10 16S rDNA and rhizobium leguminosarum Rhizobium
Leguminosarum strain INTA D156 16S rRNA genes (Sequence ID:KX066064.1 similitude)
Up to 99%.
2.3rd, physiological and biochemical property is identified
With reference to《Common bacteria system identification handbook》(east show pearl, the wonderful English common bacterias system identification handbook Beijing of Cai:Section
Learn publishing house, 2011.) and《Microbiology Experiment》(Shen Ping, Fan Xiurong, Li Guang force Microbiology Experiment (third edition) Beijing:
Higher Education Publishing House, 1999.) determine bacterial strain H10 physiological and biochemical property.As a result it is chmosynthetic heterotrophs, energy to show bacterial strain H10
Salt by the use of various carbohydrate and organic acid is as carbon source, if utilizing glucose, lactose, D-ribose, D- fibers two
Sugar, D-R, mannitol, xylose, D- galactolipins, fructose, dulcitol and inositol；Ammonium salt, nitrate and majority can be utilized
Amino acid can be used as nitrogen source；The Congo red suction micro- suction color of colour response；Litmus milk reaction is non-condensing, does not produce acid；Fiber can not be utilized
Element and starch；It is unable to hydrolyzed casein；3- ketone group lactose is not utilized；Acid is produced from mannitol；It is often accompanied by containing growth on sugar culture-medium
Abundant extracellular mucus；Do not produce hydrogen sulfide；Precipitation is not produced in calcium glycerophosphate culture medium.
In view of above-mentioned form, analysis of physio biochemical characteristics and 16s rDNA sequence homology analysis results, step 1 is separated
Purify obtained bacterial strain H10 and be accredited as rhizobium leguminosarum (Rhizobium leguminosarum).The rhizobium leguminosarum
(Rhizobium leguminosarum) H10 is preserved in Chinese microorganism strain preservation management committee on December 14th, 2015
(abbreviation CGMCC, address is member's meeting common micro-organisms center：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number is
CGMCC No.11878, hereinafter referred to as rhizobium leguminosarum H10.Rhizobium leguminosarum H10 is on November 28th, 2016 by this Shen
Applicant please proposes Chinese invention patent application, Application No. 201611072530.2.
The preparation of comparative example 2, rhizobium leguminosarum H10 microbial inoculums
1st, rhizobium leguminosarum H10 inclined-plane culture
The rhizobium leguminosarum H10 of picking comparative example 1 is inoculated in solid medium and carries out inclined-plane culture, and 56 are cultivated at 28 DEG C
Hour, obtain the rhizobium leguminosarum H10 of inclined-plane culture.
2nd, the activation of strain
The rhizobium leguminosarum H10 of picking step 1 inclined-plane culture, is inoculated in 500mL fluid nutrient mediums, at 28 DEG C
Culture 72 hours, obtains rhizobium leguminosarum H10 bacterium solutions.
3rd, seed tank culture
The rhizobium leguminosarum H10 bacterium solutions of 3L steps 2 are taken, are accessed in 100L seeding tanks (containing 60L fluid nutrient mediums)
Seed culture is carried out, shaken cultivation is cultivated for 60 hours under 30 DEG C, 120rpm, obtains rhizobium leguminosarum H10 seed liquors.
4th, fermentation tank culture
The rhizobium leguminosarum H10 seed liquors of step 3 are accessed in 10% ratio (volume ratio) and (contained in 1000L fermentation tanks
600L fluid nutrient mediums) fermented and cultured is carried out, shaken cultivation 60 hours under 28 DEG C, 150rpm obtain rhizobium leguminosarum H10 hairs
Rhizobium leguminosarum H10 content is 3,000,000,000 cfu/mL in zymotic fluid, the rhizobium leguminosarum H10 zymotic fluids.
5th, the preparation of rhizobium leguminosarum H10 microbial inoculums
Crushed after turf is spontaneously dried, cross 100 mesh sieves, then adjusted pH value to be 6.8 with limewash, gone out at 121 DEG C
Bacterium 60min, obtains sterile turf.
The rhizobium leguminosarum H10 zymotic fluids of step 4 and the sterile turf are mixed, then the Multiplying culture under the conditions of 28 DEG C
48h, obtains rhizobium leguminosarum H10 microbial inoculums, packing storage.Finished product is qualified after testing, the pea in the rhizobium leguminosarum H10 microbial inoculums
Rhizobium H10 content is 2.0 × 108Cfu/ grams.
The preparation of comparative example 3, rhizobium leguminosarum ACCC16505 microbial inoculums
1st, rhizobium leguminosarum ACCC16505 inclined-plane culture
Picking rhizobium leguminosarum ACCC16505 is inoculated in solid medium and carries out inclined-plane culture, 56 is cultivated at 28 DEG C small
When, obtain the rhizobium leguminosarum ACCC16505 of inclined-plane culture.
2nd, the activation of strain
The rhizobium leguminosarum ACCC16505 of picking step 1 inclined-plane culture, is inoculated in 500mL fluid nutrient mediums,
Cultivated 72 hours at 28 DEG C, obtain rhizobium leguminosarum ACCC16505 bacterium solutions.
3rd, seed tank culture
The rhizobium leguminosarum ACCC16505 bacterium solutions of 3L steps 2 are taken, are accessed in 100L seeding tanks (containing the training of 60L liquid
Support base) seed culture is carried out, shaken cultivation is cultivated for 60 hours under 30 DEG C, 120rpm, obtains rhizobium leguminosarum ACCC16505 kinds
Sub- liquid.
4th, fermentation tank culture
The rhizobium leguminosarum ACCC16505 seed liquors of step 3 are accessed in 1000L fermentation tanks in 10% ratio (volume ratio)
(containing 600L fluid nutrient mediums) carries out fermented and cultured, and shaken cultivation 60 hours, obtain rhizobium leguminosarum under 28 DEG C, 150rpm
Rhizobium leguminosarum ACCC16505 content is 3,000,000,000 in ACCC16505 zymotic fluids, the rhizobium leguminosarum ACCC16505 zymotic fluids
cfu/mL。
5th, the preparation of rhizobium leguminosarum ACCC16505 microbial inoculums
Crushed after turf is spontaneously dried, cross 100 mesh sieves, then adjusted pH value to be 6.8 with limewash, gone out at 121 DEG C
Bacterium 60min, obtains sterile turf.
The rhizobium leguminosarum ACCC16505 zymotic fluids of step 4 and the sterile turf are mixed, then bred under the conditions of 28 DEG C
48h is cultivated, rhizobium leguminosarum ACCC16505 microbial inoculums, packing storage is obtained.Finished product is qualified after testing, the rhizobium leguminosarum
The content of rhizobium leguminosarum ACCC16505 in ACCC16505 microbial inoculums is 2.0 × 108Cfu/ grams.
Embodiment 4, rhizobium leguminosarum m1-10-3 and rhizobium leguminosarum H10 and rhizobium leguminosarum ACCC16505 inoculation hair leaves
The Comparision Test of sweet potato kind
Select Turkmenistan hair sweet potato, Qinghai sweet potato and blue or green sweet potato one these three villose vetch kinds progress promotion growth experiment.
This experiment uses RANDOMIZED BLOCK DESIGN, is randomly provided 12 treatment regions, and each treatment region sets three cell repetitions.12
Individual treatment region is respectively m1-10-3- Qinghai sweet potato treatment region, m1-10-3- Turkmenistan hair sweet potato treatment region and the blue or green sweet potatos one of m1-10-3-
These three rhizobium leguminosarum m1-10-3 microbial inoculum treatment regions of number treatment region, H10- Qinghai sweet potato treatment region, H10- Turkmenistan Mao Tiaochu
Manage the blue or green number treatment region of sweet potato of area and H10- these three rhizobium leguminosarum H10 microbial inoculum treatment regions, the processing of ACCC16505- Qinghai sweet potato
Area, ACCC16505- Turkmenistan hair sweet potato treatment region and the treatment region of blue or green sweet potatos of ACCC16505- one these three rhizobium leguminosarums
ACCC16505 microbial inoculum treatment regions, control-Qinghai sweet potato treatment region, control-Turkmenistan hair sweet potato treatment region and control-green grass or young crops sweet potato one
These three Bu Jiejun control treatments areas for the treatment of region.
Each cell size is 4 ㎡, soil pH 8.3, Indigenous Rhizobia par 2.0 × 102Cfu/g dry ground.According to
Villose vetch application rate is 7.5kg/hm2Prepare seed.
M1-10-3- Qinghai sweet potato treatment region, m1-10-3- Turkmenistan hair sweet potato treatment region, m1-10-3- number processing of blue or green sweet potato
M1-10-3- Qinghai sweet potato seed, m1-10-3- Turkmenistan hair sweet potato seed, number seed of the blue or green sweet potatos of m1-10-3- are sowed respectively by area (will
The rhizobium leguminosarum m1-10-3 microbial inoculums of embodiment 2 respectively with Qinghai sweet potato seed, Turkmenistan hair sweet potato seed and blue or green number seed of sweet potato
According to 1：10 mass ratio mixing, mix thoroughly obtained seed be referred to as m1-10-3- Qinghai sweet potato seed, m1-10-3- soil
Number seed of the blue or green sweet potato of Ku Manmao sweet potatos seed, m1-10-3-).
H10- Qinghai sweet potato treatment region, H10- Turkmenistan hair sweet potato treatment region, the blue or green number treatment region of sweet potato of H10- are sowed respectively
H10- Qinghai sweet potato seed, H10- Turkmenistan hair sweet potato seed, the blue or green number seed of sweet potato of H10- are (by the rhizobium leguminosarum H10 of comparative example 2
Microbial inoculum is respectively with Qinghai sweet potato seed, Turkmenistan hair sweet potato seed and blue or green number seed of sweet potato according to 1：10 mass ratio is mixed, mixed
Even obtained seed is referred to as H10- Qinghai sweet potato seed, H10- Turkmenistan hair sweet potato seed, number seed of the blue or green sweet potatos of H10-).
ACCC16505- Qinghai sweet potato treatment region, ACCC16505- Turkmenistan hair sweet potato treatment region, the blue or green sweet potatos one of ACCC16505-
It is blue or green that number treatment region sows ACCC16505- Qinghai sweet potato seed, ACCC16505- Turkmenistan hair sweet potato seed, ACCC16505- respectively
Number seed of sweet potato (by the rhizobium leguminosarum ACCC16505 microbial inoculums of comparative example 3 respectively with Qinghai sweet potato seed, Turkmenistan hair sweet potato kind
Son and blue or green number seed of sweet potato are according to 1：10 mass ratio mixing, mix obtained seed thoroughly and be referred to as ACCC16505- Qinghai sweet potato
Sub- seed, ACCC16505- Turkmenistan hair sweet potato seed, number seed of the blue or green sweet potatos of ACCC16505-).
Control-Qinghai sweet potato treatment region, control-Turkmenistan hair sweet potato treatment region, control-treatment region of green grass or young crops sweet potato one are sowed respectively
CK- Qinghai sweet potato seed, CK- Turkmenistan hair sweet potato seed and CK- number seed of blue or green sweet potato (are distinguished the sterile turf in embodiment 2
With Qinghai sweet potato seed, Turkmenistan hair sweet potato seed and blue or green number seed of sweet potato according to 1：10 mass ratio mixes, mixes what is obtained thoroughly
Seed is referred to as CK- Qinghai sweet potato seed, number seed of CK- Turkmenistan hair sweet potato seed and the blue or green sweet potatos of CK-).
Above-mentioned seed is uniformly seeded into soil according to Quarter Design, type of seeding is drilling, line-spacing is 10cm.It is above-mentioned
Each treatment region is in addition to the seed sowed is different, and other conditions are identical.After planting cultivate under the same conditions to florescence,
Record each treatment region each above-ground plant parts height (plant height), aerial part fresh weight and under ground portion fresh weight and obtain plant
Fresh weight (aerial part fresh weight and under ground portion fresh weight sum), and the average value of each group is calculated, respectively obtain plant mean height
Degree, plant mean fresh.
Table 2, the plant average height of each treatment region and plant mean fresh
Table 3, rhizobium leguminosarum m1-10-3 microbial inoculums and rhizobium leguminosarum ACCC16505 microbial inoculums and rhizobium leguminosarum H10 microbial inoculums
Growth promotion comparative effectiveness
Note：M1-10-3 represents rhizobium leguminosarum m1-10-3 microbial inoculum treatment regions, and H10 is represented at rhizobium leguminosarum H10 microbial inoculums
Area is managed, ACCC16505 represents rhizobium leguminosarum ACCC16505 microbial inoculum treatment regions, and control represents Bu Jiejun control treatments area.
As a result as shown in table 2 and table 3, show：
1st, Turkmenistan hair sweet potato phase of Turkmenistan hair sweet potato of rhizobium leguminosarum m1-10-3 microbial inoculums processing with not connecing bacterium control treatment
Than plant height adds 62.36%, and plant fresh weight adds 98.39%；The Qinghai sweet potato of rhizobium leguminosarum m1-10-3 microbial inoculums processing
Son is compared with the Qinghai sweet potato for not connecing bacterium control treatment, and plant height adds 45.05%, and plant fresh weight adds 6.68%；Pea
The blue or green sweet potato one of rhizobium m1-10-3 microbial inoculums processing is compared with the blue or green sweet potato one for not connecing bacterium control treatment, and plant height is added
56.07%, plant fresh weight adds 14.22%；
2nd, Turkmenistan hair sweet potato and rhizobium leguminosarum ACCC16505 microbial inoculums of rhizobium leguminosarum m1-10-3 microbial inoculums processing are handled
Turkmenistan hair sweet potato compare, plant height adds 51.36%, and plant fresh weight adds 55.55%；Rhizobium leguminosarum m1-10-3 bacterium
The Qinghai sweet potato of agent processing is compared with the Qinghai sweet potato that rhizobium leguminosarum ACCC16505 microbial inoculums are handled, and plant height is added
19.89%, plant fresh weight adds -8.41%；The blue or green sweet potato one of rhizobium leguminosarum m1-10-3 microbial inoculums processing and rhizobium leguminosarum
The blue or green sweet potato one of ACCC16505 microbial inoculums processing is compared, and plant height adds 40.10%, and plant fresh weight adds 53.33%；
3rd, Turkmenistan hair sweet potato of rhizobium leguminosarum m1-10-3 microbial inoculums processing and the Tu Ku of rhizobium leguminosarum H10 microbial inoculums processing
Graceful hair sweet potato is compared, and plant height adds 36.69%, and plant fresh weight adds 32.34%；The processing of rhizobium leguminosarum m1-10-3 microbial inoculums
Qinghai sweet potato compared with the Qinghai sweet potato that rhizobium leguminosarum H10 microbial inoculums are handled, plant height adds 2.14%, the increase of plant fresh weight
4.96%；The blue or green sweet potato one that the blue or green sweet potato one of rhizobium leguminosarum m1-10-3 microbial inoculums processing is handled with rhizobium leguminosarum H10 microbial inoculums
Compare, plant height adds 4.72%, and plant fresh weight adds 6.03%.
The above results show rhizobium leguminosarum m1-10-3 of the invention to villose vetch than rhizobium leguminosarum H10 and pea
Rhizobium ACCC16505 has more significant promotion growth and effect of increasing production.
<110>INST OF AGRICULTURAL RESOURCES
<120>Rhizobium and its application that one plant of promotion villose vetch increases
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1364
<212> DNA
<213>Rhizobium leguminosarum (Rhizobium leguminosarum)
<400> 1
tgcgctacca tgcaagtcga gcgcgtagca atacgagcgg cagacgggtg agtaacgcgt 60
gggaatctac ccttgactac ggaataacgc agggaaactt gtgctaatac cgtatgtgtc 120
cttcgggaga aagatttatc ggtcaaggat gagcccgcgt tggattagct agttggtggg 180
gtaaaggcct accaaggcga cgatccatag ctggtctgag aggatgatca gccacattgg 240
gactgagaca cggcccaaac tcctacggga ggcagcagtg gggaatattg gacaatgggc 300
gcaagcctga tccagccatg ccgcgtgagt gatgaaggcc ctagggttgt aaagctcttt 360
caccggagaa gataatgacg gtatccggag aagaagcccc ggctaacttc gtgccagcag 420
ccgcggtaat acgaaggggg ctagcgttgt tcggaattac tgggcgtaaa gcgcacgtag 480
gcggatcgat cagtcagggg tgaaatccca gggctcaacc ctggaactgc ctttgatact 540
gtcgatctgg agtatggaag aggtgagtgg aattccgagt gtagaggtga aattcgtaga 600
tattcggagg aacaccagtg gcgaaggcgg ctcactggtc cattactgac gctgaggtgc 660
gaaagcgtgg ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgaat 720
gttagccgtc gggcagtata ctgttcggtg gcgcagctaa cgcattaaac attccgcctg 780
gggagtacgg tcgcaagatt aaaactcaaa ggaattgacg ggggcccgca caagcggtgg 840
agcatgtggt ttaattcgaa gcaacgcgca gaaccttacc agcccttgac atgcccggct 900
acttgcagag atgcaaggtt cccttcgggg accgggacac aggtgctgca tggctgtcgt 960
cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct cgcccttagt 1020
tgccagcatt gagttgggca ctctaagggg actgccggtg ataagccgag aggaaggtgg 1080
ggatgacgtc aagtcctcat ggcccttacg ggctgggcta cacacgtgct acaatggtgg 1140
tgacagtggg cagcgagcac gcgagtgtga gctaatctcc aaaagccatc tcagttcgga 1200
ttgcactctg caactcgagt gcatgaagtt ggaatcgcta gtaatcgcgg atcagcatgc 1260
cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccatgg gagttggttt 1320
tacccgaagg tagtgcgcta accgcaagga ggcagcaacc gcga 1364
Claims (10)
1. rhizobium leguminosarum (Rhizobium leguminosarum), its bacterial strain number is m1-10-3, it is in China Microbiological bacterium
The deposit number for planting preservation administration committee common micro-organisms center is CGMCC No.11877.
2. promote the microbial inoculum of villose vetch growth, it is characterised in that：The microbial inoculum contains the rhizobium leguminosarum described in claim 1
Or/and the metabolin of the rhizobium leguminosarum.
3. microbial inoculum according to claim 2, it is characterised in that：The microbial inoculum for improve villose vetch individual plant weight and/or
Improve the microbial inoculum of villose vetch plant height.
4. described in the rhizobium leguminosarum (Rhizobium leguminosarum) or Claims 2 or 3 described in claim 1
Application of the microbial inoculum in the product for promoting villose vetch growth is prepared.
5. application according to claim 4, it is characterised in that：It is single that the promotion villose vetch is grown to raising villose vetch
Plant weight amount and/or raising villose vetch plant height.
6. the biological organic fertilizer containing the microbial inoculum described in Claims 2 or 3.
7. described in the rhizobium leguminosarum (Rhizobium leguminosarum) or Claims 2 or 3 described in claim 1
Application of the biological organic fertilizer in villose vetch growth is promoted described in microbial inoculum or claim 6.
8. application according to claim 7, it is characterised in that：It is single that the promotion villose vetch is grown to raising villose vetch
Plant weight amount and/or raising villose vetch plant height.
9. the method for the rhizobium leguminosarum (Rhizobium leguminosarum) described in claim 1 is cultivated, including by right
It is required that the rhizobium leguminosarum (Rhizobium leguminosarum) described in 1 is cultivated in the culture medium for cultivating rhizobium
The step of.
10. the method for microbial inoculum described in Claims 2 or 3 is prepared, including by the rhizobium leguminosarum described in claim 1
(Rhizobium leguminosarum) and/or the metabolin of the rhizobium leguminosarum (Rhizobium leguminosarum)
As the microbial inoculum active component the step of.
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| CN115261249A (en) * | 2022-02-24 | 2022-11-01 | 中交路桥北方工程有限公司 | Pea rhizobium and application thereof |
| CN115261249B (en) * | 2022-02-24 | 2023-08-04 | 中交路桥建设有限公司 | Rhizobium pisum and application thereof |
| CN115927088A (en) * | 2022-10-28 | 2023-04-07 | 中国中医科学院中药研究所 | Rhizobium ginseng and application thereof |
| CN115927088B (en) * | 2022-10-28 | 2023-11-10 | 中国中医科学院中药研究所 | Ginseng rhizobium and application thereof |
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