CN107164261B - One plant of rhizobium for promoting villose vetch to increase and its application - Google Patents
One plant of rhizobium for promoting villose vetch to increase and its application Download PDFInfo
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- CN107164261B CN107164261B CN201710354501.3A CN201710354501A CN107164261B CN 107164261 B CN107164261 B CN 107164261B CN 201710354501 A CN201710354501 A CN 201710354501A CN 107164261 B CN107164261 B CN 107164261B
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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 rhizobium leguminosarum for promoting villose vetch to increase the invention discloses one plant and its application.The bacterial strain number of the rhizobium leguminosarum is m1-10-3, 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 are dramatically increased with the plant height of rhizobium leguminosarum ACCC16505, plant fresh weight than not meeting 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 rhizobium for promoting villose vetch to increase the present invention relates to one plant in field of agricultural microorganism and its application.
Background technique
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 good green
Fertile crop is mainly distributed on China the Yellow River, Huaihe River, one band of Haihe basin, 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 short time subzero 20 DEG C of low temperature, and seedling
Wintering rate it is very high, while villose vetch is drought-enduring and impoverishment tolerant is also very strong, generally 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 determine to implement since summer nineteen ninety-five in the whole nation
It is the villose vetch cultivated area that green manure crop is again legume to manufacture " fertile_soil plan " of the organic fertilizer as main contents
It is being continuously increased.The research and application of Rhizobium leguminosarum worldwide have more than 100 years history, almost in whole world model
It is 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 one kind can infect leguminous plant root
(minority is stem) forms the bacterium that root nodule carries out biological nitrogen fixation, and rhizobium with legume symbiosis system are made in biological nitrogen fixation
With strongest system, the nitrogen-fixing microorganism in soil converts the nitrogen in air in the ammonia of plant available, the nitrogen fixed
About the 65% of biological nitrogen fixation total amount.Currently, leguminous plant is planted in degenerated soil to have been to be concerned by more and more people, because
Nitrogen nutrition is poorer in degenerated soil, and soil fertilizer can be improved by the symbiotic azotification of leguminous plant and rhizobium
Power, Rhizobium Inoculation can be improved yield and the nitrogen reserves of leguminous plant.If the leguminous plant of plantation is without corresponding high
Imitate nitrogen of the Rhizobium strains therewith in symbiosis dross and fixed air, the reference state nitrogen that they will be completely dependent in soil, not only
The nitrogen that can be consumed in soil instead cannot be supplemented, soil fertility is caused to decline.Therefore, in order to give full play to rhizobium and pulse family
The symbiotic azotification of plant needs to screen high efficient strain and carries out artificial infection to maintain soil nitrogen balance.It develops and utilizes
Leguminous plant and the potentiality of rhizobium Symbiotic nitrogen-fixing effect are very big.During biological nitrogen fixation, according to rhizobium to host plant
Specificity, be suitable for same rhizobium nodulation and nitrogen fixation plant be classified as family, rhizobium can turn between each plant in race
It connects, referred to as cross inoculation group.It is above cross inoculation group that villose vetch and pea are applied in rhizobium, and rhizobium leguminosarum can both be inoculated with not
It can also be inoculated with different cultivars pea with villose vetch, but effect of inoculation differs greatly.
There is data to suggest that the nitrogen fixing capacity of villose vetch is highest between different green manure types.While villose vetch
The yield increasing effect of Soil building effect and green manuring, stub land is all very significant.Nearly ten years, China villose vetch cultivated area
Constantly expand, the cultivation of villose vetch and is gradually increased using technology, the research and application of villose vetch Rhizobium Inoculation technology
It is enlarged attention.
Summary of the invention
The technical problem to be solved by the present invention is to how remarkably promote villose vetch to increase.
In order to solve the above technical problems, the present invention provides one plant of rhizobium.
Rhizobium provided by the present invention are rhizobium leguminosarum (Rhizobium leguminosarum), and bacterial strain number is
M1-10-3, the bacterial strain are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on December 14th, 2015
Center (abbreviation CGMCC, address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number are CGMCC No.11877,
Hereinafter referred to as rhizobium leguminosarum m1-10-3.
Rhizobium leguminosarum m1-10-3 belongs to Gram-negative, short and small rod-shaped, and thallus dyeing is uneven, formed coloring with
The ring bodies of not colored part, non-staining part lipid content are higher.Without gemma, has the raw flagellum in end or peritrichous, can transport
It is dynamic.Thallus size is (0.5-0.8) × (1.1-2.9) micron.It is grown on yeast juice mannite agar culture base plane, bacterium colony
Rounded protrusion, neat in edge is smooth compared with wetted surface, and quality is uniform, light milky white, and lawn is sticky.Rhizobium leguminosarum m1-
10-3 has the 16S rDNA sequence of sequence 1 in sequence table.
In order to solve the above technical problems, the present invention also provides the microbial inoculums for promoting villose vetch growth.
The microbial inoculum provided by the present invention for promoting villose vetch growth, contains rhizobium leguminosarum m1-10-3 or/and pea root
The metabolin of tumor bacterium m1-10-3.
The microbial inoculum for promoting villose vetch growth concretely improves villose vetch single plant weight and/or improves hair leaf sweet potato
The microbial inoculum of sub- plant height.The villose vetch single plant weight refer to villose vetch single plant aerial part and under ground portion weight it
With.
The active constituent of above-mentioned microbial inoculum can be the metabolism of rhizobium leguminosarum m1-10-3 or/and rhizobium leguminosarum m1-10-3
Object, the active constituent of above-mentioned microbial inoculum can also contain other biological ingredient or abiotic component, the other active components of above-mentioned microbial inoculum
Those skilled in the art can determine villose vetch Plant weight and/or plant height facilitation effect according to microbial inoculum.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 stalk, loose shell, straw, peanut shell, corn flour, bean powder, starch, turf and the animal of all kinds of crops
At least one of excrement；The liquid-carrier can be water；In the microbial inoculum, rhizobium leguminosarum m1-10-3 or/and pea root
The metabolin of tumor bacterium m1-10-3 can be with the fermentation liquid of the living cells, living cells that are cultured, the filtrate of cell culture or cell
Exist with the form of the mixture of filtrate.The dosage form of the microbial inoculum can be a variety of dosage forms, as liquor, emulsion, suspending agent, pulvis,
Granule, wettable powder or water dispersible granules.
Wherein, the metabolin of rhizobium leguminosarum m1-10-3 is that rhizobium leguminosarum m1-10-3 ferments to train in microbial liquid
Support the substance cultivated in base.
As needed, surfactant (such as polysorbas20, Tween 80), adhesive, stabilization can be also added in the microbial inoculum
Agent (such as antioxidant), pH adjusting agent.
Rhizobium leguminosarum m1-10-3 or described promotes the microbial inoculum of villose vetch growth to promote villose vetch growth in preparation
Application in product and the application in promotion villose vetch growth all belong to the scope of protection of the present invention.
In above-mentioned application, the promotion villose vetch growth can be raising villose vetch single plant weight and/or raising hair leaf
Sweet potato plant height.
In above-mentioned application, microbial inoculum or contain that the product for promoting villose vetch growth can grow for promotion villose vetch
The bio-feritlizer of the microbial inoculum for promoting villose vetch growth.
In the application, the villose vetch can be Turkmenistan hair sweet potato, Qinghai sweet potato, and/or green sweet potato No.1.
In order to solve the above technical problems, the present invention also provides a kind of methods for cultivating rhizobium leguminosarum m1-10-3.
The method of culture rhizobium leguminosarum m1-10-3 provided by the present invention, including in the culture for cultivating rhizobium
The step of rhizobium leguminosarum m1-10-3 is cultivated in base.
The culture medium for cultivating 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 are settled to 1000ml with water, adjust
PH value is 6.8-7.0, and sterilizing obtains the culture medium for cultivating rhizobium.
It is demonstrated experimentally that rhizobium leguminosarum m1-10-3 of the invention is 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, practicability with higher and replicability to villose vetch, be expected to develop into villose vetch
A new excellent combination technique in rhizobium application field.The present invention is in villose vetch planting industry with wide before
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 abbreviation: CGMCC
Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date: on December 14th, 2015
Collection is registered on the books number: CGMCC No.11877
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.Experimental method in following embodiments is unless otherwise specified
Conventional method.The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
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 agricultural microorganism center (abbreviation ACCC, address: Beijing sea the moon
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 China Committee for Culture Collection of Microorganisms agricultural microorganism center from collection day.Pea root nodule
Bacterium (Rhizobium leguminosarum) ACCC 16505 hereinafter abbreviation rhizobium leguminosarum ACCC16505.
The preparation 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 are settled to 1000ml with water, and tune pH value is 6.8-7.0,121 DEG C of sterilizing 30min.
The preparation 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 is settled to 1000ml with 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
1, the separation of bacterial strain
Wild villose vetch root nodule is acquired from Hai Dongshi safety area, Qinghai Province, (takes mannitol 10g, yeast extract with plate
1g, dipotassium hydrogen phosphate 0.5g, calcium sulfate 0.2g, magnesium sulfate 0.2g, sodium chloride 0.1g, the ammonium molybdate that mass content is 1% are water-soluble
Liquid 1ml and mass content are 1% boric acid aqueous solution 1ml, 0.5% Congo red 1ml, 20g agar are settled to 1L, pH value with water
For the isolated bacterial strain m1-10-3 of 6.8-7.0) scribing line.
2, the identification of bacterial strain
2.1, Morphological Identification
It will be in logarithmic growth phase, and bacterium colony size is stablized, the bacterial strain m1-10-3 that above-mentioned steps 1 are separated and purified
Single colonie state description is carried out, main includes size, color, transparency, wettability, the bacterium colony surface state, bacterium colony side of bacterium colony
Edge state.On the other hand, to the bacterial strain m1-10-3 for being in logarithmic growth phase, optical microphotograph sem observation is used after smear staining
The form of thallus.
The result shows that bacterial strain m1-10-3 belongs to Gram-negative, short and small rod-shaped, thallus dyeing is uneven, forms coloring
With the ring bodies of not colored part, non-staining part lipid content is higher.Without gemma, have the raw flagellum in end or peritrichous, energy
Movement.Thallus size is (0.5-0.8) × (1.1-2.9) micron.It is grown on yeast juice mannite agar culture base plane, bacterium
Rounded protrusion is fallen, neat in edge is smooth compared with wetted surface, and quality is uniform, light milky white, and lawn is sticky.
2.2,16S rDNA sequence homology analysis
Using the 16S rDNA segment of the resulting bacterial strain m1-10-3 of colony polymerase chain reaction (PCR) method amplification step 1, related reagent is by complete
Formula King Company provides.To 16S rRNA genetic fragment expand and cloning and sequencing the result shows that, the 16S of bacterial strain m1-10-3
RDNA has the nucleotide sequence of sequence 1 in sequence table.The rRNA genetic fragment of bacterial strain m1-10-3 and rhizobium leguminosarum
(Rhizobium leguminosarum) bacterial strain CCBAU 85022 16S rRNA Gene Partial sequence (Sequence ID:
EU256422.1 similitude) is up to 99%.
2.3, physiological and biochemical property is identified
With reference to " common bacteria system identification handbook " (east show pearl, Beijing Cai Miaoying common bacteria system identification handbook: section
Publishing house, 2011.) and " Microbiology Experiment " (Beijing Shen Ping, Fan Xiurong, Li Guangwu Microbiology Experiment (third edition):
Higher Education Publishing House, 1999.) physiological and biochemical property of measurement bacterial strain m1-10-3.The result shows that bacterial strain m1-10-3 is to change energy
Heterotroph, can be using the salt of various carbohydrate and organic acid as carbon source, as can utilizing glucose, lactose, D-ribose, D-
Cellobiose, D-arabinose, mannitol, xylose, D- galactolipin, fructose, dulcitol and inositol；Ammonium salt, nitrate can be utilized
With most amino acid as nitrogen source；The Congo red micro- suction color of suction colour response；Litmus milk reaction is non-condensing, does not produce acid；It cannot utilize
Cellulose and starch；It is unable to hydrolyzed casein；3- ketone group lactose is not utilized；Acid is produced from mannitol；Normal containing growth on sugar culture-medium
With extracellular mucus abundant；Do not generate hydrogen sulfide；Precipitating is not generated in calcium glycerophosphate culture medium.
In view of above-mentioned form, analysis of physio biochemical characteristics and 16s rDNA sequence homology analysis as a result, step 1 is separated
It purifies obtained bacterial strain m1-10-3 and is 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
Reason committee common micro-organisms center (abbreviation CGMCC, address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), preservation are 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 inoculum
1, the inclined-plane culture of rhizobium leguminosarum m1-10-3
The rhizobium leguminosarum m1-10-3 of picking embodiment 1 is inoculated in solid medium and carries out inclined-plane culture, trains at 28 DEG C
It supports 56 hours, obtains the rhizobium leguminosarum m1-10-3 of inclined-plane culture.
2, the activation of strain
The rhizobium leguminosarum m1-10-3 of picking step 1 inclined-plane culture, is inoculated in 500mL fluid nutrient medium, 28
It is cultivated 48 hours at DEG C, obtains rhizobium leguminosarum m1-10-3 bacterium solution.
3, seed tank culture
The rhizobium leguminosarum m1-10-3 bacterium solution for taking 3L step 2, is accessed in 100L seeding tank (containing 60L Liquid Culture
Base) seed culture is carried out, it cultivates within shaken cultivation 56 hours at 30 DEG C, 120rpm, obtains rhizobium leguminosarum m1-10-3 seed
Liquid.
4, fermentation tank culture
It will be in the rhizobium leguminosarum m1-10-3 seed liquor access 1000L fermentor of step 3 in 10% ratio (volume ratio)
(containing 600L fluid nutrient medium) carries out fermented and cultured, and shaken cultivation 56 hours, obtain rhizobium leguminosarum at 28 DEG C, 150rpm
M1-10-3 fermentation liquid, the content of rhizobium leguminosarum m1-10-3 is 3,000,000,000 cfu/mL in the rhizobium leguminosarum m1-10-3 fermentation liquid.
5, the preparation of rhizobium leguminosarum m1-10-3 microbial inoculum
It crushes, sieves with 100 mesh sieve after turf is spontaneously dried, be then 6.8 with limewash tune pH value, go out at 121 DEG C
Bacterium 60min obtains sterile turf.
The rhizobium leguminosarum m1-10-3 fermentation liquid of step 4 and the sterile turf are mixed, then are proliferated training under the conditions of 28 DEG C
48h is supported, rhizobium leguminosarum m1-10-3 microbial inoculum, packing storage are obtained.Qualified, rhizobium leguminosarum m1-10-3 bacterium through detection finished product
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, is made M type, intermediate recess is according to the size system of seedling
At V-type aperture, the height of M type filter paper is that the length of test tube subtracts 4cm.Test tube used is 2.0cm × 20cm.
Prepared nitrogen-free nutrient solution is respectively charged into the test tube with filter paper supporter, height is located at H-type filter paper
Recess, each processing repeats 4 test tubes, and sets control tube 4, after being sealed after filling with high-temperature and high-presure resistent plastic film,
15 pounds of 121 DEG C of sterilizing 1h are spare.
Uniform Turkmenistan hair sweet potato seed is selected, uses 0.1%HgCl after impregnating 5min with 95% ethyl alcohol2Surface sterilizing
5min, then with aseptic water washing 10 times.The seed of disinfection is placed in 28-30 DEG C of incubators and keeps the temperature vernalization.Illumination is required from top
Irradiation, root are protected from light, can place 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 is set to be repeated three times, duplicate every time
Experimental method is as follows:
Rhizobium leguminosarum m1-10-3 processing: 10 successful seeds of vernalization are put into bacteria suspension and (are diluted with fluid nutrient medium
The content of the rhizobium leguminosarum m1-10-3 fermentation liquid of 2 step 4 of embodiment to rhizobium leguminosarum m1-10-3 are 2.0 × 107cfu/
ML obtains bacteria suspension) in impregnate 30min, with sterile tweezers carefully press from both sides out seed be put into it is fixed in the filter paper bridge of water planting liquid test tube
Good sowing, bacteria suspension is averagely sucked in test tube.After sealing in lighting box illumination cultivation, illumination is irradiated from top, when illumination
Between 14h/10h, root is protected from light, and intensity of illumination is 7000lux -8000lux；Temperature: 25-30 DEG C.
Do not connect bacterium control treatment: the difference with rhizobium leguminosarum m1-10-3 processing, which is only that, replaces with liquid for bacteria suspension
Culture medium, other operations are identical.
Table 1, Turkmenistan hair sweet potato are inoculated with rhizobium leguminosarum m1-10-3 water culture experiment result
The results are shown in Table 1, shows after rhizobium leguminosarum m1-10-3 to be inoculated with to Turkmenistan hair sweet potato, plant height, plant root
Long, plant fresh weight and plant weights are not than connecing bacterium control growth by 32.56%, 18.01%, 19.32% and 47.62% respectively, table
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
1, the separation of bacterial strain
Wild pea root nodule is acquired from Qinghai Province's Ledu County, (takes mannitol 10g, yeast extract 1g, phosphoric acid hydrogen two with plate
Potassium 0.5g, calcium sulfate 0.2g, magnesium sulfate 0.2g, sodium chloride 0.1g, the ammonium molybdate aqueous solution 1ml and quality that mass content is 1%
The boric acid aqueous solution 1ml that content is 1%, 0.5% Congo red 1ml, 20g agar are settled to 1L, pH value 6.8-7.0 with water) it draws
The isolated bacterial strain H10 of line.
2, the identification of bacterial strain
2.1, Morphological Identification
It will be in logarithmic growth phase, and bacterium colony size is stablized, above-mentioned steps 1 separate and the bacterial strain H10 purified is carried out
Single colonie state description, main includes size, color, transparency, wettability, the bacterium colony surface state, colony edge shape of bacterium colony
State.On the other hand, to the bacterial strain H10 for being in logarithmic growth phase, the shape of optical microphotograph sem observation thallus is used after smear staining
State.
The result shows that bacterial strain H10 belongs to Gram-negative, small rod-short, thallus 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 the raw flagellum in end or peritrichous, movement 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.Extracellular mucus abundant is being often accompanied by containing the growth-gen on sugar culture-medium.
2.2,16S rDNA sequence homology analysis
Using the 16S rDNA segment of the resulting bacterial strain H10 of colony polymerase chain reaction (PCR) method amplification step 1, related reagent is by Quan Shijin
Company provides.To 16S rRNA genetic fragment expand and cloning and sequencing the result shows that, the 16S rDNA of bacterial strain H10 has
The nucleotide sequence of sequence 2 in sequence table.The 16S rDNA and rhizobium leguminosarum Rhizobium of bacterial strain H10
The similitude of the 16S rRNA gene (Sequence ID:KX066064.1) of leguminosarum strain INTA D156
Up to 99%.
2.3, physiological and biochemical property is identified
With reference to " common bacteria system identification handbook " (east show pearl, Beijing Cai Miaoying common bacteria system identification handbook: section
Publishing house, 2011.) and " Microbiology Experiment " (Beijing Shen Ping, Fan Xiurong, Li Guangwu Microbiology Experiment (third edition):
Higher Education Publishing House, 1999.) physiological and biochemical property of measurement bacterial strain H10.The result shows that bacterial strain H10 is chmosynthetic heterotrophs, energy
Using the salt of various carbohydrate and organic acid as carbon source, as can utilizing glucose, lactose, D-ribose, D- fiber two
Sugar, D-arabinose, mannitol, xylose, D- galactolipin, fructose, dulcitol and inositol；Ammonium salt, nitrate and majority can be utilized
Amino acid can be used as nitrogen source；The Congo red micro- suction color of suction colour response；Litmus milk reaction is non-condensing, does not produce acid；Fiber cannot be utilized
Element and starch；It is unable to hydrolyzed casein；3- ketone group lactose is not utilized；Acid is produced from mannitol；It grows and is often accompanied by containing sugar culture-medium
Extracellular mucus abundant；Do not generate hydrogen sulfide；Precipitating is not generated in calcium glycerophosphate culture medium.
In view of above-mentioned form, analysis of physio biochemical characteristics and 16s rDNA sequence homology analysis as a result, step 1 is separated
It purifies obtained bacterial strain H10 and is accredited as rhizobium leguminosarum (Rhizobium leguminosarum).The rhizobium leguminosarum
(Rhizobium leguminosarum) H10 has been preserved in Chinese microorganism strain preservation management committee on December 14th, 2015
Member's meeting common micro-organisms center (abbreviation CGMCC, address are as follows: 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, and application No. is 201611072530.2.
The preparation of comparative example 2, rhizobium leguminosarum H10 microbial inoculum
1, the inclined-plane culture of rhizobium leguminosarum H10
The rhizobium leguminosarum H10 of picking comparative example 1 is inoculated in solid medium and carries out inclined-plane culture, cultivates 56 at 28 DEG C
Hour, obtain the rhizobium leguminosarum H10 of inclined-plane culture.
2, the activation of strain
The rhizobium leguminosarum H10 of picking step 1 inclined-plane culture, is inoculated in 500mL fluid nutrient medium, at 28 DEG C
Culture 72 hours, obtains rhizobium leguminosarum H10 bacterium solution.
3, seed tank culture
The rhizobium leguminosarum H10 bacterium solution for taking 3L step 2, is accessed in 100L seeding tank (containing 60L fluid nutrient medium)
Seed culture is carried out, cultivates within shaken cultivation 60 hours at 30 DEG C, 120rpm, obtains rhizobium leguminosarum H10 seed liquor.
4, fermentation tank culture
(it will contain in the rhizobium leguminosarum H10 seed liquor access 1000L fermentor of step 3 in 10% ratio (volume ratio)
600L fluid nutrient medium) fermented and cultured is carried out, shaken cultivation 60 hours at 28 DEG C, 150rpm obtain rhizobium leguminosarum H10 hair
Zymotic fluid, the content of rhizobium leguminosarum H10 is 3,000,000,000 cfu/mL in the rhizobium leguminosarum H10 fermentation liquid.
5, the preparation of rhizobium leguminosarum H10 microbial inoculum
It crushes, sieves with 100 mesh sieve after turf is spontaneously dried, be then 6.8 with limewash tune pH value, go out at 121 DEG C
Bacterium 60min obtains sterile turf.
The rhizobium leguminosarum H10 fermentation liquid of step 4 and the sterile turf are mixed, then Multiplying culture under the conditions of 28 DEG C
48h obtains rhizobium leguminosarum H10 microbial inoculum, packing storage.It is qualified through detection finished product, the pea in the rhizobium leguminosarum H10 microbial inoculum
The content of rhizobium H10 is 2.0 × 108Cfu/ grams.
The preparation of comparative example 3, rhizobium leguminosarum ACCC16505 microbial inoculum
1, the inclined-plane culture of rhizobium leguminosarum ACCC16505
Picking rhizobium leguminosarum ACCC16505 is inoculated in solid medium and carries out inclined-plane culture, and it is small that 56 are cultivated at 28 DEG C
When, obtain the rhizobium leguminosarum ACCC16505 of inclined-plane culture.
2, the activation of strain
The rhizobium leguminosarum ACCC16505 of picking step 1 inclined-plane culture, is inoculated in 500mL fluid nutrient medium,
It is cultivated 72 hours at 28 DEG C, obtains rhizobium leguminosarum ACCC16505 bacterium solution.
3, seed tank culture
The rhizobium leguminosarum ACCC16505 bacterium solution for taking 3L step 2 is accessed in 100L seeding tank and (is trained containing 60L liquid
Support base) seed culture is carried out, it cultivates within shaken cultivation 60 hours at 30 DEG C, 120rpm, obtains rhizobium leguminosarum ACCC16505 kind
Sub- liquid.
4, fermentation tank culture
It will be in the rhizobium leguminosarum ACCC16505 seed liquor access 1000L fermentor of step 3 in 10% ratio (volume ratio)
(containing 600L fluid nutrient medium) carries out fermented and cultured, and shaken cultivation 60 hours, obtain rhizobium leguminosarum at 28 DEG C, 150rpm
ACCC16505 fermentation liquid, the content of rhizobium leguminosarum ACCC16505 is 3,000,000,000 in the rhizobium leguminosarum ACCC16505 fermentation liquid
cfu/mL。
5, the preparation of rhizobium leguminosarum ACCC16505 microbial inoculum
It crushes, sieves with 100 mesh sieve after turf is spontaneously dried, be then 6.8 with limewash tune pH value, go out at 121 DEG C
Bacterium 60min obtains sterile turf.
The rhizobium leguminosarum ACCC16505 fermentation liquid of step 4 and the sterile turf are mixed, then are proliferated under the conditions of 28 DEG C
48h is cultivated, rhizobium leguminosarum ACCC16505 microbial inoculum, packing storage are obtained.Qualified, rhizobium leguminosarum through detection finished product
The content of rhizobium leguminosarum ACCC16505 in ACCC16505 microbial inoculum is 2.0 × 108Cfu/ grams.
Embodiment 4, rhizobium leguminosarum m1-10-3 and rhizobium leguminosarum H10 and rhizobium leguminosarum ACCC16505 are inoculated with hair leaf
The Comparision Test of sweet potato kind
Selection Turkmenistan hair sweet potato, Qinghai sweet potato and green these three villose vetch kinds of sweet potato No.1 carry out promotion growth experiment.
This test uses RANDOMIZED BLOCK DESIGN, is randomly provided 12 treatment regions, and each treatment region sets three cells and repeats.12
A treatment region is respectively the Qinghai m1-10-3- sweet potato treatment region, the Turkmenistan m1-10-3- hair sweet potato treatment region and m1-10-3- blueness sweet potato one
These three rhizobium leguminosarum m1-10-3 microbial inoculum treatment regions of number treatment region, the Qinghai H10- sweet potato treatment region, the Turkmenistan H10- Mao Tiaochu
Manage area and H10- blueness sweet potato No.1 treatment region these three rhizobium leguminosarum H10 microbial inoculum treatment regions, the processing of the Qinghai ACCC16505- sweet potato
Area, the Turkmenistan ACCC16505- hair sweet potato treatment region and ACCC16505- blueness sweet potato No.1 treatment region these three rhizobium leguminosarums
ACCC16505 microbial inoculum treatment region, control-Qinghai sweet potato treatment region, control-Turkmenistan hair sweet potato treatment region and control-blueness sweet potato No.1
These three Bu Jiejun control treatment 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.
The Qinghai m1-10-3- sweet potato treatment region, the Turkmenistan m1-10-3- hair sweet potato treatment region, the processing of m1-10-3- blueness sweet potato No.1
The Qinghai m1-10-3- sweet potato seed, the Turkmenistan m1-10-3- hair sweet potato seed, m1-10-3- blueness sweet potato No.1 seed are sowed respectively by area (will
The rhizobium leguminosarum m1-10-3 microbial inoculum of embodiment 2 respectively with Qinghai sweet potato seed, Turkmenistan hair sweet potato seed and green sweet potato No.1 seed
The Qinghai m1-10-3- sweet potato seed, m1-10-3- soil are referred to as according to the mass ratio mixing of 1:10, the seed mixed thoroughly
Ku Manmao sweet potato seed, m1-10-3- blueness sweet potato No.1 seed).
The Qinghai H10- sweet potato treatment region, the Turkmenistan H10- hair sweet potato treatment region, H10- blueness sweet potato No.1 treatment region are sowed respectively
The Qinghai H10- sweet potato seed, the Turkmenistan H10- hair sweet potato seed, H10- blueness sweet potato No.1 seed are (by the rhizobium leguminosarum H10 of comparative example 2
Microbial inoculum is mixed according to the mass ratio of 1:10 with Qinghai sweet potato seed, Turkmenistan hair sweet potato seed and green sweet potato No.1 seed respectively, is mixed
Even obtained seed is referred to as the Qinghai H10- sweet potato seed, the Turkmenistan H10- hair sweet potato seed, H10- blueness sweet potato No.1 seed).
The Qinghai ACCC16505- sweet potato treatment region, the Turkmenistan ACCC16505- hair sweet potato treatment region, ACCC16505- blueness sweet potato one
It is green that number treatment region sows the Qinghai ACCC16505- sweet potato seed, the Turkmenistan ACCC16505- hair sweet potato seed, ACCC16505- respectively
Sweet potato No.1 seed (by the rhizobium leguminosarum ACCC16505 microbial inoculum of comparative example 3 respectively with Qinghai sweet potato seed, Turkmenistan hair sweet potato kind
Son and green sweet potato No.1 seed are referred to as the Qinghai ACCC16505- sweet potato according to the mass ratio mixing of 1:10, the seed mixed thoroughly
Sub- seed, the Turkmenistan ACCC16505- hair sweet potato seed, ACCC16505- blueness sweet potato No.1 seed).
Control-Qinghai sweet potato treatment region, control-Turkmenistan hair sweet potato treatment region, control-blueness sweet potato No.1 treatment region are sowed respectively
The Qinghai CK- sweet potato seed, the Turkmenistan CK- hair sweet potato seed and CK- blueness sweet potato No.1 seed (are distinguished the sterile turf in embodiment 2
With Qinghai sweet potato seed, Turkmenistan hair sweet potato seed and green sweet potato No.1 seed according to the mass ratio mixing of 1:10, mix thoroughly
Seed is referred to as the Qinghai CK- sweet potato seed, the Turkmenistan CK- hair sweet potato seed and CK- blueness sweet potato No.1 seed).
Above-mentioned seed is uniformly seeded into soil according to Quarter Design, type of seeding is drilling, line-spacing 10cm.It is above-mentioned
For each treatment region in addition to the seed sowed is different, other conditions are identical.It after planting cultivates under the same conditions to florescence,
It records each above-ground plant parts height (plant height) of each treatment region, aerial part fresh weight and under ground portion fresh weight and obtains plant
Fresh weight (the sum of aerial part fresh weight and under ground portion fresh weight), 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 inoculum and rhizobium leguminosarum ACCC16505 microbial inoculum and rhizobium leguminosarum H10 microbial inoculum
Growth promotion comparative effectiveness
Note: m1-10-3 indicates that rhizobium leguminosarum m1-10-3 microbial inoculum treatment region, H10 indicate at rhizobium leguminosarum H10 microbial inoculum
Area is managed, ACCC16505 indicates rhizobium leguminosarum ACCC16505 microbial inoculum treatment region, and control indicates Bu Jiejun control treatment area.
As a result as shown in table 2 and table 3, show:
1, Turkmenistan hair sweet potato of rhizobium leguminosarum m1-10-3 microbial inoculum processing and Turkmenistan hair sweet potato phase for not connecing bacterium control treatment
Than plant height increases 62.36%, and plant fresh weight increases 98.39%；The Qinghai sweet potato of rhizobium leguminosarum m1-10-3 microbial inoculum processing
Son is compared with the Qinghai sweet potato for not connecing bacterium control treatment, and plant height increases 45.05%, and plant fresh weight increases 6.68%；Pea
Compared with the green sweet potato No.1 for not connecing bacterium control treatment, plant height increases the green sweet potato No.1 of rhizobium m1-10-3 microbial inoculum processing
56.07%, plant fresh weight increases 14.22%；
2, Turkmenistan hair sweet potato and rhizobium leguminosarum ACCC16505 microbial inoculum of rhizobium leguminosarum m1-10-3 microbial inoculum processing are handled
Turkmenistan hair sweet potato compare, plant height increases 51.36%, and plant fresh weight increases 55.55%；Rhizobium leguminosarum m1-10-3 bacterium
Compared with the Qinghai sweet potato that rhizobium leguminosarum ACCC16505 microbial inoculum is handled, plant height increases the Qinghai sweet potato of agent processing
19.89%, plant fresh weight increases -8.41%；The green sweet potato No.1 of rhizobium leguminosarum m1-10-3 microbial inoculum processing and rhizobium leguminosarum
The green sweet potato No.1 of ACCC16505 microbial inoculum processing is compared, and plant height increases 40.10%, and plant fresh weight increases 53.33%；
3, the Tu Ku of Turkmenistan hair sweet potato of rhizobium leguminosarum m1-10-3 microbial inoculum processing and the processing of rhizobium leguminosarum H10 microbial inoculum
Graceful hair sweet potato is compared, and plant height increases 36.69%, and plant fresh weight increases 32.34%；The processing of rhizobium leguminosarum m1-10-3 microbial inoculum
Qinghai sweet potato compared with the Qinghai sweet potato that rhizobium leguminosarum H10 microbial inoculum is handled, plant height increases 2.14%, and plant fresh weight increases
4.96%；The green sweet potato No.1 of rhizobium leguminosarum m1-10-3 microbial inoculum processing and the green sweet potato No.1 of rhizobium leguminosarum H10 microbial inoculum processing
It compares, plant height increases 4.72%, and plant fresh weight increases 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>one plants of rhizobium for promoting villose vetch to increase and its application
<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), bacterial strain number is m1-10-3, in China Microbiological bacterium
The deposit number of kind preservation administration committee common micro-organisms center is CGMCC No.11877.
2. promoting the microbial inoculum of villose vetch growth, it is characterised in that: the microbial inoculum contains pea root nodule described in claim 1
Bacterium.
3. microbial inoculum according to claim 2, it is characterised in that: the microbial inoculum be improve villose vetch single plant weight and/or
Improve the microbial inoculum of villose vetch plant height.
4. rhizobium leguminosarum described in claim 1(Rhizobium leguminosarum) or it is described in claim 2 or 3
Application of the microbial inoculum in the product that preparation promotes villose vetch growth.
5. application according to claim 4, it is characterised in that: the promotion villose vetch, which is grown to, improves villose vetch list
Plant weight amount and/or raising villose vetch plant height.
6. containing the biological organic fertilizer of microbial inoculum described in claim 2 or 3.
7. rhizobium leguminosarum described in claim 1(Rhizobium leguminosarum) or it is described in claim 2 or 3
Microbial inoculum or biological organic fertilizer as claimed in claim 6 are promoting the application in villose vetch growth.
8. application according to claim 7, it is characterised in that: the promotion villose vetch, which is grown to, improves villose vetch list
Plant weight amount and/or raising villose vetch plant height.
9. cultivating rhizobium leguminosarum described in claim 1(Rhizobium leguminosarum) method, including by right
It is required that rhizobium leguminosarum described in 1(Rhizobium leguminosarum) cultivated in the culture medium for cultivating rhizobium
The step of.
10. the method for preparing microbial inoculum described in Claims 2 or 3, including by rhizobium leguminosarum described in claim 1(Rhizobium leguminosarum) active constituent as the microbial inoculum the step of.
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| CN107815428B (en) * | 2017-11-14 | 2020-06-09 | 四川农业大学 | Cadmium-removing rhizobium KG2, microbial inoculum containing rhizobium and application of microbial inoculum |
| CN107904192B (en) * | 2017-12-26 | 2020-06-02 | 四川农业大学 | Rhizobium V9-2 and application thereof |
| CN107904193B (en) * | 2017-12-26 | 2020-06-02 | 四川农业大学 | Rhizobium V14-2 and application thereof |
| CN109182194B (en) * | 2018-09-27 | 2021-12-24 | 中国农业科学院农业资源与农业区划研究所 | Rhizobium oridonii for promoting growth of corolla dentiger and culture method and application thereof |
| CN113854318B (en) * | 2021-11-12 | 2023-05-12 | 中国农业科学院农业资源与农业区划研究所 | Application of Pityrosporum indicum in improving drought resistance of Mao She seeds |
| CN113999804B (en) * | 2021-12-02 | 2023-01-24 | 四川省烟草公司凉山州公司 | Rhizobium SCAUY033 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 |
| 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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