CN110283751A - The microbial inoculum and its bacterial strain uses therefor that promote leguminous plant to grow and application - Google Patents
The microbial inoculum and its bacterial strain uses therefor that promote leguminous plant to grow and application Download PDFInfo
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- CN110283751A CN110283751A CN201910619650.7A CN201910619650A CN110283751A CN 110283751 A CN110283751 A CN 110283751A CN 201910619650 A CN201910619650 A CN 201910619650A CN 110283751 A CN110283751 A CN 110283751A
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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
- 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
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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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- 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
Abstract
The invention discloses the microbial inoculum for promoting leguminous plant growth and its bacterial strain uses therefor and applications.The bacterial strain is rhizobium leguminosarum, and bacterial strain number is H10, is CGMCC No.11878 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 and plant weights are compared with plant weights and are dramatically increased with plant height, the plant fresh weight of inoculation rhizobium leguminosarum ACCC16001 than not connecing bacterium control after pea and bean seed inoculation rhizobium leguminosarum H10 CGMCC No.11878.Present invention practicability with higher and replicability are expected to develop into a new excellent combination technique in rhizobium leguminosarum application field.The present invention has broad application prospects in leguminous plant planting industry.
Description
It is that November 28, invention and created name in 2016 are that the application, which is application No. is the 201611072530.2, applying date,
The divisional application of " one plant of rhizobium leguminosarum and its fermentation culture method and application ".
Technical field
The present invention relates to the microbial inoculum for promoting leguminous plant growth in field of agricultural microorganism and its bacterial strain uses therefor and applications.
Background technique
Pea (Pisum sativum L.) is that pulse family Pisum is annual or more year raw herbaceous plant, seed are edible
With cauline leaf is good green manure and feed, and is a kind of crop widely distributed in the world, because such because its adaptability is very wide
It plants and utilizes area larger, 621.43 ten thousand hectares, 955.82 ten thousand tons of total yield of the dry pea cultivated area in the whole world；Whole world green pea kind
224.13 ten thousand hectares of area are planted, 1697.50 ten thousand tons of total yield, be the fourth-largest legume crop in the world.The dry pea cultivated area in China
94.00 ten thousand hectares, 119.00 ten thousand tons of total yield；129.59 ten thousand hectares of green pea cultivated area, 1027.43 ten thousand tons of total yield.China is dry
Pea cultivated area and total yield account for global 15.13% and 12.45% respectively, and green pea cultivated area and total yield account for entirely respectively
The 57.82% of the world and 60.53%, it is only second to Canada, is second-biggest-in-the-world pea producing country, is accounted in the production of world's pea
There is very important status.Semen viciae fabae (Vicia faba Linn) is pulse family Vetch annual herb, be grain, vegetables and
Feed, green manure dual-purpose crop.Semen viciae fabae nutritive value is abundant, contains 8 kinds of essential amino acids, and carbohydrate content is 47%~
60%, edible, also capable of using as feed, green manure and nectariferous plant plantation, are grain, vegetables and feed, green manure dual-purpose crop.
Nitrogen is one of most important nutrient in plant growth, and crops are obtained dependent on application chemical nitrogen fertilizer
Volume increase is actually will be in air as the nitrogen-fixing microorganism acquired by cost, and in soil to consume the energy and pollution environment
The process that nitrogen is converted into the ammonia of plant available is efficient and environmental-friendly.Rhizobium (rhizobia) are a kind of extensive
The gramnegative bacterium being present in soil.They can form symbiosis with leguminous plant, by infecting leguminous plant
Root or stem formed root nodule or (and) stem tumor, the nitrogen in air is then converted to plant available in the form of homobium
High-quality nitrogen-the glutamic acid and paddy ammonia phenol amine substance utilized.The research and application of Rhizobium leguminosarum, which have more than 100 years, goes through
History is almost all advocated in worldwide and carries out legume inoculation to legume.Rhizobium and legume symbiosis system
It is one of known strongest system of biological nitrogen fixation ability, annual amount of nitrogen fixation accounts for 65% or more of the total amount of nitrogen fixation of global biology.Full generation
Boundary leguminous plant about 748 belongs to, and 20,000 kinds, it has been known that there is 172 categories in China, 1,485 kinds, goes back
There are many constantly can be included and record with the leguminous plant of rhizobium symbiosis dross.In these leguminous plants only
There is the kind less than 16% to do the investigation of dross, and only 0.3%-0.5% studied symbiosis.Therefore it develops
It is very big with utilization leguminous plant and the potentiality of rhizobium Symbiotic nitrogen-fixing effect.During biological nitrogen fixation, according to rhizobium to posting
The plant for being suitable for same rhizobium nodulation and nitrogen fixation is classified as family by the specificity of main plant, and rhizobium are in race between each plant
It can transfer, referred to as cross inoculation group.Rhizobium leguminosarum can be used for Pisum, Vicia, Lathyrus and sweet potato inoculation.Plantation
When legume, symbiotic azotification and grain yield is can be improved in the suitable rhizobium of adaptation to local conditions inoculation.Pea and bean
With drought resisting, barren-resistant, in China western part, there is special adaptability in soil depletion, the serious area of desertification, and passes through it
Biological nitrogen fixation inhibits soil degradation and saves nitrogenous fertilizer usage amount, is conducive to cultivate soil fertility, repairs ecology damage.In recent years
Come, China's pea and bean cultivated area constantly expands, and the cultivation and popularization of pea and bean, pea and silkworm are attached great importance in various regions
The research and application of beans Rhizobium Inoculation technology are also constantly enlarged attention.
Summary of the invention
The technical problem to be solved by the present invention is to how dramatically increase leguminous plant Plant weight and/or plant height.
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
H10, the bacterial strain are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 14th, 2015
(abbreviation CGMCC, address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number are CGMCC No.11878, below
Referred to as rhizobium leguminosarum H10.
The small rod-short of rhizobium leguminosarum H10, thallus size is 0.5~0.9 micron × 1.2~6.0 microns, in different cultures
The thalli morphology of basal growth is different.Typically contain Poly-β-hydroxybutyric Acid salt particle.Without gemma, have the raw flagellum in end or peritrichous,
Movement, it is aerobic.25~30 DEG C of optimum growth temperature, optimal pH 6.0~7.0.Bacterium colony is rounded, neat in edge, protrusion, and colourless half
Transparent or light milky white, it is sticky.Diameter 2 after being grown 3-6 days on yeast juice mannitol inorganic salts agar medium plate~
4mm.Extracellular mucus abundant is being often accompanied by containing the growth-gen on sugar culture-medium.
In order to solve the above technical problems, the present invention also provides the microbial inoculums for promoting leguminous plant growth.
The microbial inoculum provided by the present invention for promoting leguminous plant growth, contains rhizobium leguminosarum H10 or/and rhizobium leguminosarum
The metabolin of H10.
The microbial inoculum for promoting leguminous plant growth, which concretely improves leguminous plant single plant weight and/or improves pulse family, plants
The microbial inoculum of object plant height.The leguminous plant single plant weight refers to the aerial part weight of leguminous plant single plant.
The active constituent of above-mentioned microbial inoculum can be the metabolin of rhizobium leguminosarum H10 or/and rhizobium leguminosarum H10, above-mentioned bacterium
The active constituent of agent can also contain other biological ingredient or abiotic component, the other active components art technology of above-mentioned microbial inoculum
Personnel can determine leguminous plant Plant weight and/or plant height facilitation effect according to microbial inoculum.
The microbial inoculum may also include carrier.The carrier can be solid carrier or liquid-carrier.The solid carrier is mine
Object material, biomaterial；The mineral material can for turf, clay, talcum, kaolin, montmorillonite, white carbon, zeolite, silica and
At least one of diatomite；The biomaterial be the stalks of all kinds of crops, loose shell, straw, peanut shell, corn flour, bean powder,
At least one of starch, turf and excrement of animal；The liquid-carrier can be water；In the microbial inoculum, rhizobium leguminosarum H10
Or/and the metabolin of rhizobium leguminosarum H10 can be with the filter of the fermentation liquid of the living cells, living cells that are cultured, cell culture
The form of liquid or cell and the mixture of filtrate exists.The dosage form of the microbial inoculum can be a variety of dosage forms, such as liquor, emulsion, suspension
Agent, pulvis, granule, wettable powder or water dispersible granules.
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.
The product that rhizobium leguminosarum H10 or described promotes the microbial inoculum of leguminous plant growth to promote leguminous plant to grow in preparation
In application and promote leguminous plant growth in application all belong to the scope of protection of the present invention.
In above-mentioned application, the promotion leguminous plant growth can be raising leguminous plant single plant weight and/or raising pulse family
Plant plant height.
In above-mentioned application, microbial inoculum or contain that the product for promoting leguminous plant growth can grow for promotion leguminous plant
The bio-feritlizer of the microbial inoculum for promoting leguminous plant growth.
In the application, the leguminous plant can be pea or semen viciae fabae.
In order to solve the above technical problems, the present invention also provides a kind of methods for cultivating rhizobium leguminosarum H10.
The method of culture rhizobium leguminosarum H10 provided by the present invention, including in the culture medium for cultivating rhizobium
The step of cultivating rhizobium leguminosarum H10.
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 being planted after rhizobium leguminosarum H10 is inoculated with grassland 23, grassland 24, grassland 28, Qinghai 12 and Qinghai 13
Plant height degree, plant fresh weight and plant weights increase than not connecing bacterium control fresh weight: plant mean fresh is not than connecing bacterium adjoining tree point
Not Zeng Chang by 76.09%, 46.67%, 78.95%, 89.12% and 76.10%, plant mean height ratio does not connect bacterium adjoining tree point
Not Zeng Chang by 109.19%, 29.60%, 69.97%, 127.31% and 90.81%, plant average dry weight ratio does not connect bacterium adjoining tree
Increase by 556.61%, 455.00%, 597.16%, 875.00% and 377.78% (table 1 and 3) respectively.Pea and bean seed
Plant height, plant fresh weight after inoculation rhizobium leguminosarum (Rhizobium leguminosarum) H10 CGMCC No.11878
It compares and dramatically increases with plant weights than being inoculated with plant height, the plant fresh weight of rhizobium leguminosarum ACCC16001 with plant weights
(table 4): grassland 23, grassland 24, grassland 28, Qinghai 12 and Qinghai 13 plant average height increase separately 70.25%,
6.19%, 47.08%, 61.13% and 63.49%, plant mean fresh increases separately 58.51%, 41.94%, 63.86%,
78.92% and 55.21%, plant average dry weight increases separately 482.05%, 256.79%, 526.02%, 748.83% and
340.27% (table 4).Show rhizobium leguminosarum of the invention (Rhizobium leguminosarum) H10 CGMCC
No.11878 has more significant promotion growth and effect of increasing production to pea and bean than rhizobium leguminosarum ACCC16001.This hair
Bright practicability with higher and replicability are expected to develop into a new excellent combination in rhizobium leguminosarum application field
Technology.The present invention has broad application prospects in leguminous plant (such as pea and bean) planting industry.
Preservation explanation
Strain name: rhizobium leguminosarum Rhizobium leguminosarum
Strain number: H10
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.11878
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 16001 in following embodiments was in 1991 1
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 16001 hereinafter abbreviation rhizobium leguminosarum ACCC16001.
Embodiment 1, rhizobium leguminosarum (Rhizobium leguminosarum) H10 CGMCC 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 1 in sequence table.The 16S rDNA and rhizobium leguminosarum Rhizobium of bacterial strain H10
Leguminosarum strain INTA D156 16S rRNA gene (Sequence ID:KX066064.1) similitude
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.
Embodiment 2, the microbial inoculum for promoting leguminous plant growth
1, the inclined-plane culture of rhizobium leguminosarum H10
Rhizobium leguminosarum H10 is inoculated in solid medium and carries out inclined-plane culture, cultivates 56h at 28 DEG C.
The preparation method of solid medium is as follows: glycerine 5ml, mannitol 5g, yeast extract 1g, K2HPO40.5g, nothing
Water MgSO40.2g, CaSO4·2H2O 0.2g, NaCl 0.1g, 1% (NH4)6Mo7O24·4H2O 1ml, 1%H3BO31ml,
Agar 20g is settled to 1000ml with water, and tune pH value is 6.8-7.0,121 DEG C of sterilizing 30min.
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
72h is cultivated, rhizobium leguminosarum H10 bacterium solution is obtained.
The preparation method of fluid nutrient medium is as follows: glycerine 5ml, mannitol 5g, yeast extract 1g, K2HPO40.5g, nothing
Water MgSO40.2g, CaSO4·2H2O 0.2g, NaCl 0.1g, 1% (NH4)6Mo7O24·4H2O 1ml, 1%H3BO31ml,
It is settled to 1000ml with water, tune pH value is 6.8-7.0,121 DEG C of sterilizing 30min.
3, seed tank culture
The rhizobium leguminosarum H10 bacterium solution for taking 6L step 2, is accessed in 100L seeding tank the (liquid containing 60L step 2
Culture medium) seed culture is carried out, shaken cultivation 60h is cultivated 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)
The fluid nutrient medium of 600L step 2) fermented and cultured is carried out, shaken cultivation 60h, obtains rhizobium leguminosarum at 28 DEG C, 150rpm
H10 fermentation liquid, 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 in the ratio of 3:1, then in 28 DEG C of conditions
Lower Multiplying culture 48h obtains rhizobium leguminosarum H10 microbial inoculum, packing storage.Qualified, rhizobium leguminosarum H10 bacterium through detection finished product
The content of rhizobium leguminosarum H10 in agent is 2.0 × 108Cfu/ grams.
Embodiment 3, rhizobium leguminosarum (Rhizobium leguminosarum) H10 CGMCC No.11878 and pea root
The Comparision Test of tumor bacterium ACCC16001 inoculation Foreign Banks' Entries
One, the preparation of ACCC16001 microbial inoculum
1, the inclined-plane culture of rhizobium leguminosarum ACCC16001
The rhizobium leguminosarum ACCC16001 solid medium for being inoculated in embodiment 2 is subjected to inclined-plane culture, is trained at 28 DEG C
Support 56h.
2, the activation of strain
The rhizobium leguminosarum ACCC16001 of picking step 1 inclined-plane culture is inoculated in the liquid training of 500mL embodiment 2
It supports in base, cultivates 72h at 28 DEG C, obtain rhizobium leguminosarum ACCC16001 bacterium solution.
3, seed tank culture
The rhizobium leguminosarum ACCC16001 bacterium solution for taking 6L step 2, is accessed in 100L seeding tank (containing 60L embodiment
2 fluid nutrient medium) seed culture is carried out, shaken cultivation 60h is cultivated at 30 DEG C, 120rpm, obtains rhizobium leguminosarum
ACCC16001 seed liquor.
4, fermentation tank culture
It will be in the rhizobium leguminosarum ACCC16001 seed liquor access 1000L fermentor of step 3 in 10% ratio (volume ratio)
(fluid nutrient medium containing 600L embodiment 2) carries out fermented and cultured, and shaken cultivation 60h, obtains pea at 28 DEG C, 150rpm
Rhizobium ACCC16001 fermentation liquid, the content of rhizobium leguminosarum ACCC16001 in the rhizobium leguminosarum ACCC16001 fermentation liquid
For 3,000,000,000 cfu/ml.
5, the preparation of rhizobium leguminosarum ACCC16001 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 ACCC16001 fermentation liquid of step 4 and the sterile turf are mixed in the ratio of 3:1, then 28
Multiplying culture 48h under the conditions of DEG C obtains rhizobium leguminosarum ACCC16001 microbial inoculum, packing storage.Qualified, pea through detection finished product
The content of rhizobium leguminosarum H10 in beans rhizobium ACCC16001 microbial inoculum is 2.0 × 108cfu/g。
Two, promote pea and bean growth experiment
Select grassland 23, grassland 24 and this 3 Foreign Banks' Entries of grassland 28 and Qinghai 12 and the two Vicia Fabas of Qinghai 13
Carry out promotion growth experiment:
Uniform seed is selected, after being impregnated 2-3 minutes with the concentrated sulfuric acid for several times with sterile water washing, to go deacidification to be
Only, the seed that obtains that treated.By treated seed the sets respectively H10-kind processing, ACCC16001-kind of each kind
Processing handles (not connecing bacterium control treatment) with bacterium-varietal check is not connect.
The rhizobium leguminosarum H10 microbial inoculum of embodiment 2 and seed are mixed according to the mass ratio of 1:10 in H10-kind processing
It closes, mix thoroughly.By the rhizobium leguminosarum H10 microbial inoculum of embodiment 2 and grassland 23, grassland 24,13 seed of grassland 28, Qinghai 12 and Qinghai
H10-grassland 23, H10-grassland 24, H10-grass are referred to as according to the mass ratio mixing of 1:10, the seed mixed thoroughly
Original 28, H10-Qinghai 12 and H10-Qinghai 13.
ACCC16001 microbial inoculum and seed are mixed according to the mass ratio of 1:10 in ACCC16001-kind processing, mixed thoroughly.
ACCC16001 microbial inoculum and grassland 23, grassland 24, grassland 28, Qinghai 12 and 13 seed of Qinghai are mixed according to the mass ratio of 1:10
The seed close, mixed thoroughly is referred to as ACCC16001-grassland 23, ACCC16001-grassland 24, ACCC16001-grassland
28, ACCC16001-Qinghai 12 and ACCC16001-Qinghai 13.
Do not connect in the processing of bacterium-varietal check by embodiment 2 sterile turf and seed mix according to the mass ratio of 1:10
It closes, mix thoroughly.By in embodiment 2 sterile turf and grassland 23, grassland 24, grassland 28, Qinghai 12 and 13 seed of Qinghai according to
The mass ratio mixing of 1:10, the seed mixed thoroughly are referred to as CK-grassland 23, CK-grassland 24, CK-grassland 28, CK-
Qinghai 12 and CK-Qinghai 13.Bacterium-will not be connect with above-mentioned by above-mentioned H10-kind processing, ACCC16001-kind processing
The seed of varietal check processing is seeded into the basin equipped with identical soil respectively (equipped with 2.5kg soil in the basin of 15cm × 18cm
Earth), every basin sows 10 seeds.Experiment in triplicate, repeats 4 basins of each processing every time.Greenhouse is after planting put in the same terms
Lower culture records each above-ground plant parts height (plant height), aerial part fresh weight (plant fresh weight) and ground to florescence
Upper part dry weight (plant weights), and calculate the average value of each group, respectively obtain plant average height, plant mean fresh and
Plant average dry weight.
The result shows that rhizobium leguminosarum ACCC16001 is inoculated with kind grassland 23, grassland 24, grassland 28, Qinghai 12 and blueness
Behind sea 13, plant height, plant fresh weight and plant weights increase than not connecing bacterium control fresh weight: plant mean fresh ratio does not connect bacterium pair
Increase by 11.09%, 3.33%, 9.21%, 5.70% and 13.46% respectively according to plant, plant average dry weight is not than connecing bacterium control
Plant increases by 12.81%, 55.56%, 11.36%, 14.86% and 8.52% respectively, and plant mean height ratio does not connect bacterium control
Plant increases by 22.87%, 22.05%, 15.56%, 41.07% and 16.71% (table 1 and 2) respectively.By rhizobium leguminosarum H10
After being inoculated with grassland 23, grassland 24, grassland 28, Qinghai 12 and Qinghai 13, plant height, plant fresh weight and plant weights ratio do not connect bacterium
Control fresh weight increases: plant mean fresh increases by 76.09% than not connecing bacterium adjoining tree respectively, 46.67%, 78.95%,
89.12% and 76.10%, plant average dry weight increases by 556.61% than not connecing bacterium adjoining tree respectively, 455.00%,
597.16%, 875.00% and 377.78%, plant mean height ratio do not connect bacterium adjoining tree increase by 109.19% respectively,
29.60%, 69.97%, 127.31% and 90.81% (table 1 and 3).Pea and bean seed is inoculated with rhizobium leguminosarum
Plant height, plant fresh weight and plant weights ratio connect after (Rhizobium leguminosarum) H10 CGMCC No.11878
Plant height, the plant fresh weight of kind rhizobium leguminosarum ACCC16001 is compared with plant weights to be dramatically increased (table 4): grassland 23, grass
Original 24, grassland 28, Qinghai 12 and Qinghai 13 plant average height increase separately 70.25%, 6.19%, 47.08%,
61.13% and 63.49%, plant mean fresh increases separately 58.51%, 41.94%, 63.86%, 78.92% and
55.21%, plant average dry weight increases separately 482.05%, 256.79%, 526.02%, 748.83% and 340.27% (table
4).Show rhizobium leguminosarum of the invention (Rhizobium leguminosarum) H10 CGMCC No.11878 than pea root
Tumor bacterium ACCC16001 has more significant promotion growth and effect of increasing production to pea and bean.
Table 1 does not connect bacterium-varietal check plant average height and plant mean fresh and dry weight
| Bacterium-varietal check is not connect | Plant average height (cm) | Plant mean fresh (g/ plants) | Plant average dry weight (g/10 plants) |
| The grassland CK- 23 | 28.73 | 0.460 | 0.242 |
| The grassland CK- 24 | 40.37 | 0.420 | 0.180 |
| The grassland CK- 28 | 32.77 | 0.380 | 0.176 |
| The Qinghai CK- 12 | 27.10 | 0.386 | 0.148 |
| The Qinghai CK- 13 | 28.31 | 0.431 | 0.270 |
Table 2, rhizobium leguminosarum ACCC16001 and do not connect bacterium-varietal check effect comparison result
Table 3, rhizobium leguminosarum H10 and do not connect bacterium-varietal check effect comparison result
Table 4, rhizobium leguminosarum H10 and rhizobium leguminosarum ACCC16001 effect comparison result
<110>INST OF AGRICULTURAL RESOURCES
<120>microbial inoculum and its bacterial strain uses therefor that promote leguminous plant to grow and application
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1034
<212> DNA
<213>rhizobium leguminosarum (Rhizobium leguminosarum)
<400> 1
tgatgaaggc cctagggttg taaagctctt tcaccggaga agataatgac ggtatccgga 60
gaagaagccc cggctaactt cgtgccagca gccgcggtaa tacgaagggg gctagcgttg 120
ttcggaatta ctgggcgtaa agcgcacgta ggcggatcga tcagtcaggg gtgaaatccc 180
agggctcaac cctggaactg cctttgatac tgtcgatctg gagtatggaa gaggtgagtg 240
gaattccgag tgtagaggtg aaattcgtag atattcggag gaacaccagt ggcgaaggcg 300
gctcactggt ccattactga cgctgaggtg cgaaagcgtg gggagcaaac aggattagat 360
accctggtag tccacgccgt aaacgatgaa tgttagccgt cgggcagtat actgttcggt 420
ggcgcagcta acgcattaaa cattccgcct ggggagtacg gtcgcaagat taaaactcaa 480
aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcgc 540
agaaccttac cagcccttga catgcccggc tacttgcaga gatgcaaggt tcccttcggg 600
gaccgggaca caggtgctgc atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag 660
tcccgcaacg agcgcaaccc tcgcccttag ttgccagcat tcagttgggc actctaaggg 720
gactgccggt gataagccga gaggaaggtg gggatgacgt caagtcctca tggcccttac 780
gggctgggct acacacgtgc tacaatggtg gtgacagtgg gcagcgagca cgcgagtgtg 840
agctaatctc caaaagccat ctcagttcgg attgcactct gcaactcgag tgcatgaagt 900
tggaatcgct agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac 960
acaccgcccg tcacaccatg ggagttggtt ttacccgaag gtagtgcgct aaccgcaagg 1020
aggcagctaa ccag 1034
Claims (9)
1. rhizobium leguminosarum (Rhizobium leguminosarum), bacterial strain number is H10, is protected in Chinese microorganism strain
The deposit number for hiding administration committee's common micro-organisms center is CGMCC No.11878.
2. promoting the microbial inoculum of leguminous plant growth, it is characterised in that: the microbial inoculum contains rhizobium leguminosarum described in claim 1
The metabolin of (Rhizobium leguminosarum) and the rhizobium leguminosarum (Rhizobium leguminosarum).
3. microbial inoculum according to claim 2, it is characterised in that: the microbial inoculum be improve leguminous plant single plant weight and/or
Improve the microbial inoculum of leguminous plant plant height.
4. rhizobium leguminosarum (Rhizobium leguminosarum) or described in claim 2 or 3 described in claim 1
Application of the microbial inoculum in the product that preparation promotes leguminous plant growth.
5. application according to claim 4, it is characterised in that: the promotion leguminous plant, which is grown to, improves leguminous plant list
Plant weight amount and/or raising leguminous plant plant height.
6. rhizobium leguminosarum (Rhizobium leguminosarum) or described in claim 2 or 3 described in claim 1
Microbial inoculum is promoting the application in leguminous plant growth.
7. application according to claim 6, it is characterised in that: the promotion leguminous plant, which is grown to, improves leguminous plant list
Plant weight amount and/or raising leguminous plant plant height.
8. any application in microbial inoculum according to claim 2 or 3, claim 4-7, it is characterised in that: described
Leguminous plant is pea or semen viciae fabae.
9. the method for preparing microbial inoculum described in Claims 2 or 3, including by rhizobium leguminosarum described in claim 1
(Rhizobium leguminosarum) and/or the metabolin of the rhizobium leguminosarum (Rhizobium leguminosarum)
The step of active constituent as the microbial inoculum.
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| CN109182194A (en) * | 2018-09-27 | 2019-01-11 | 中国农业科学院农业资源与农业区划研究所 | One plant of Yang Ling rhizobium for promoting coronule flower growth and its cultural method and application |
| CN114381408A (en) * | 2022-02-10 | 2022-04-22 | 青海省农林科学院 | Rhizobium fabae with drought resistance and application thereof |
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| CN106754484B (en) | 2019-08-09 |
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