CN110468073A - The slow raw rhizobium of the degeneration-resistant fixed nitrogen of one plant of suitable the Northeast and its application - Google Patents
The slow raw rhizobium of the degeneration-resistant fixed nitrogen of one plant of suitable the Northeast and its application Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/06—Coating or dressing seed
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05D—INORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C; FERTILISERS PRODUCING CARBON DIOXIDE
- C05D9/00—Other inorganic fertilisers
- C05D9/02—Other inorganic fertilisers containing trace elements
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
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- 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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- 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
Abstract
The invention discloses the slow raw rhizobium of the degeneration-resistant fixed nitrogen of one plant of suitable the Northeast and its applications.The present invention discloses slow raw rhizobium (Bradyrhizobium sp.) DBPB1 first, is CGMCC No.17502 in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center.Invention further provides the microbial inoculum comprising above-mentioned slow raw rhizobium and its applications.Slowly raw rhizobium (Bradyrhizobium sp.) DBPB1 has growth-promoting characteristic to the present invention, and there is very strong adaptability, it is bio-safety bacterial strain, the characters such as the peanut plant height of different development stage, root nodule number, ground fresh weight can be dramatically increased, greatly improve the yield of peanut, high-efficiency nitrogen-fixing is provided for one of main growing area of Chinese Peanut-the Northeast and stablizes the microorganism germ plasma resource of growth-promoting, may advantageously facilitate the green and healthy sustainable development of Chinese agriculture.
Description
Technical field
The present invention relates to microorganism fields, and in particular to the slow raw rhizobium of the degeneration-resistant fixed nitrogen of one plant of suitable the Northeast and
It is applied.
Background technique
Peanut is important one of the industrial crops and oil plant legume of China.Peanut has plantation all over China, main
The areas such as the Yellow River and Huai He River region, northeast, Guangdong and Guangxi Provinces are distributed in, Chinese Peanut production relies primarily on chemical fertilizer, and does not utilize Roots of Peanut
The effect of tumor microbial inoculum Yu peanut symbiotic nitrogen fixation this " natural nitrogenous fertilizer factory ", it is very painful.Chemical fertilizer is excessively used main
The reasons are as follows: the substitute products of 1. chemical fertilizer fall behind；2. fertilising equipment is poor, fertilizer loss is big；3. it is directed to legume,
It is completely dependent on chemical nitrogen fertilizer, causes great waste；4. for different planting system fertilizer subtract apply synergy technical research it is stagnant
Afterwards, it needs to reinforce Integration ofTechnology, innovation and application mode.Therefore, develop the environmentally protective new technology such as organic fertilizer and bio-fertilizer and
New fertilizer, reinforces Technology Integration Innovation and application is the key that China realizes that chemical fertilizer subtracts and applies synergy.
Rhizobium are the one kind of a kind of nitrogen with legume symbiosis, formation root nodule and fixed air for plant nutrient
Gram-negative bacteria.This syntaxial system has strongest nitrogen fixing capacity.In root nodule, skin of the rhizobium from leguminous plant root
Carbohydrate, mineral salt and moisture are drawn in confluent monolayer cells, are grown and are bred.They are again free in air simultaneously
Nitrogen be fixed up by nitrogen fixation, be changed into the nitrogenous compound that plant can utilize, for needed for plant life.In this way,
Rhizobium and root constitute the symbiosis to interdepend.Rhizobium secrete some organic nitrogens into soil in life process,
Root nodule can voluntarily fall off in the growth latter stage of plant, to substantially increase the fertility of soil.
Although nitragin has many advantages, such as, the dross and fixed nitrogen of rhizobium also by soil environment for example moisture, pH value,
The influence of the factors such as chemical fertilizer, pesticide.For the nitrogen fixing capacity for sending out brightness rhizobium for ensureing maximal efficiency, it is necessary to screening tool
Have and adapt to different ecological environment capacity, high-efficiency nitrogen-fixing with stronger resistance, drug resistance rhizobium strains extensively,
Just adapt to the state that Leguminosae From China effect planting range is wide, environmental difference is big, generally uses Insecticides (tech) & Herbicides (tech) and fungicide
Feelings, and have no domestic correlative study report.
Therefore, it is extremely necessary to carry out degeneration-resistant and high-efficiency nitrogen-fixing bacterial strain screening study work.
Summary of the invention
The technical problems to be solved by the invention are to provide how to carry out high-efficiency nitrogen-fixing and promote plant growth.
To solve the above problems, present invention firstly provides one plant of slow raw rhizobium.
Slowly raw rhizobium are slow raw rhizobium (Bradyrhizobium sp.) DBPB1 to the present invention, in China Microbiological
The deposit number of culture presevation administration committee common micro-organisms center is CGMCC No.17502.
Sequence 1 in the 16s rDNA sequence such as sequence table of above-mentioned slow raw rhizobium (Bradyrhizobium sp.) DBPB1
It is shown.
Slow raw rhizobium (Bradyrhizobium sp.) DBPB1 of the present invention is the degeneration-resistant fixed nitrogen for being suitble to the Northeast
Bradyrhizobium sp Arachis.
Slow raw rhizobium (Bradyrhizobium sp.) DBPB1 menses agar plate method detection of the present invention,
Hemolytic activity is feminine gender, is shown to person poultry harmless.
Slow raw rhizobium (Bradyrhizobium sp.) DBPB1 of the present invention is identified to have multiple functions feature,
It specifically includes that the function of (1) promoting growth of plants: promoting plant in the knit stitch of the plant height, root nodule number, gynophore of different development stage
The increase of number, fruit number, yield, ground fresh weight；(2) resistance: resistance to salinity is that (i.e. tolerable NaCl's is dense by 4g/100ml
Degree is 4g/100ml)；Acidproof pH6；Alkaline-resisting pH11；Drought-resistant (concentration that tolerable concentration is PEG 6000 is 30g/100ml,
Severe drought)；(3) drug resistance: the fungicide Fluoxastrobin (250mg/L) and insecticide generally used in tolerable agricultural application
The maximum of imidacloprid (58.3mg/L) uses concentration.
Invention further provides a kind of microbial inoculums.
Microbial inoculum of the present invention contains above-mentioned slow raw rhizobium (Bradyrhizobium sp.) DBPB1.
In above-mentioned microbial inoculum, the microbial inoculum has following at least one characteristics:
A1) salt tolerant；
A2) acidproof；
A3) alkaline-resisting；
A4) drought-resistant；
A5) drug resistance；
A6) fixed nitrogen；
A7) promoting growth of plants.
In above-mentioned microbial inoculum, the active constituent of the microbial inoculum can be above-mentioned slow raw rhizobium (Bradyrhizobium sp.)
DBPB1, the culture of above-mentioned slow raw rhizobium (Bradyrhizobium sp.) DBPB1 or above-mentioned slow raw rhizobium
The metabolin of (Bradyrhizobium sp.) DBPB1, the active constituent of the microbial inoculum can also containing other biological ingredient or/
And abiotic component, it is true that progress can be actually needed in other active components those skilled in the art of the microbial inoculum according to the present invention
It is fixed.
In above-mentioned microbial inoculum, other than containing active constituent, also contain auxiliary material.The auxiliary material can select as needed.
In above-mentioned microbial inoculum, the culture of slow raw rhizobium (Bradyrhizobium sp.) DBPB1 is to take root slowly
The substance in culture vessel that tumor bacterium (Bradyrhizobium sp.) DBPB1 is cultivated in microbiological culture media is such as sent out
Zymotic fluid.
In above-mentioned microbial inoculum, the metabolin of slow raw rhizobium (Bradyrhizobium sp.) DBPB1 can be from taking root slowly
It is obtained in the culture of tumor bacterium (Bradyrhizobium sp.) DBPB1.
In above-mentioned microbial inoculum, the microbiological culture media is YMA fluid nutrient medium.
In one embodiment of the invention, the microbial inoculum is specially liquid bacterial agent, is by using YMA Liquid Culture
Raw rhizobium (Bradyrhizobium sp.) DBPB1 is obtained slowly described in base culture；The slow raw rhizobium
Concentration of (Bradyrhizobium sp.) DBPB1 in the liquid bacterial agent is 1 × 1010-11cfu/ml。
The present invention further discloses above-mentioned slow raw rhizobium (Bradyrhizobium sp.) DBPB1 or above-mentioned microbial inoculum to exist
Prepare the application in the product with following at least one characteristics:
A1) salt tolerant；
A2) acidproof；
A3) alkaline-resisting；
A4) drought-resistant；
A5) drug resistance；
A6) fixed nitrogen；
A7) promoting growth of plants.
In above-mentioned application, the product can be microbial manure.
In above-mentioned microbial inoculum or application, the salt tolerant is that the concentration of the NaCl of tolerance is 4g/100ml；It is described acidproof for tolerance
PH be 6；It is described it is alkaline-resisting be tolerance pH be 11；The drought-resistant concentration for being the PEG 6000 being resistant to is 30g/100ml
(i.e. Severe drought)；The drug resistance is the fungicide Fluoxastrobin for being resistant to generally use in agricultural application and insecticide pyrrole worm
Quinoline, the maximum of the specific tolerance Fluoxastrobin are 250mg/L using concentration, and the maximum of the imidacloprid is using concentration
58.3mg/L。
The present invention further additionally provides a kind of application method of microbial inoculum.
Above-mentioned microbial inoculum application method, includes the following steps:
S1 the bacterium solution of above-mentioned slow raw rhizobium (Bradyrhizobium sp.) DBPB1) is added into microelement；
S2 the step S1) bacterium solution for obtaining addition microelement) is sprayed into vegetable seeds surface, is dried in the shade；
S3 seed dressing and coating) is carried out to the seed that step S2) dries in the shade with carboxymethylcellulose sodium solution (protective agent), is dried in the shade；
S4 it) sows.
In the application method of above-mentioned microbial inoculum, step S1) in, the slow raw rhizobium (Bradyrhizobium sp.)
The bacterium solution of DBPB1 can by using strain mother liquor that YM fluid nutrient medium culture obtains (concentration of strain mother liquor up to 1 ×
1010-11Cfu/ml obtained after) being diluted by clean tap water, the proportion of the two is 1 volume strain mother liquor: 2 volumes it is clean from
Water.
In the application method of above-mentioned microbial inoculum, step S1) in, the microelement is added in the form of liquid microelement
In bacterium solution, the content of each component of the liquid microelement are as follows: H38O32.86g/L MnSO41.81g/L CuSO4·5H2O
0.80g/L, ZnSO40.22g/L, H2MoO40.02g/L, solvent are water；The micro member added into the dilution bacterium solution
The amount of plain liquid can add liquid microelement described in 1 μ L for every 3mL bacterium solution.Wherein, the effect of the microelement is to increase battalion
It supports, improve bacterial activity and promote nodule formation and fixed nitrogen.
In the application method of above-mentioned microbial inoculum, step S2) in, the dosage of the bacterium solution is so that the surface of the seed is wet.
In the application method of above-mentioned microbial inoculum, step S3) in, the carboxymethylcellulose sodium solution (protective agent) is dense eventually
Degree is the sodium carboxymethyl cellulose solution of 10g/L.Preparation method is as follows: 10g sodium carboxymethylcellulose (800~
It 1200mPas) is dissolved in 1L deionized water, is dissolved to transparent paste glue in 60 DEG C of stirred in water bath.Described 1% carboxymethyl
The dosage of sodium cellulosate solution (protective agent) can be every kg seed 25mL, act on the attachment and seed for being to maintain microbial inoculum
Humidity.
In the application method of above-mentioned microbial inoculum, step S4) in, described the step of after planting may also include watering is being carried out,
It is to guarantee the necessary humidity of strain activity that it, which is acted on,.
Application of the application method of above-mentioned microbial inoculum in promoting growth of plants is also within protection scope of the present invention.
In the present invention, the promoting growth of plants can specifically be presented as following at least one:
B1) plant height of the plant is promoted to increase in the different development stage of the plant；
B2) the fruit number of the plant is promoted to increase in the different development stage of the plant；
B3 the root nodule number of the plant) is promoted to increase；
B4 the knit stitch number of the plant) is promoted to increase；
B5 the ground fresh weight of the plant) is promoted to increase；
B6 the yield of the plant) is promoted to increase；
The different development stage is full-bloom stage (about after planting 60 days) and/or mature harvest time (about after planting 120
It).
In above-mentioned application, the promoting growth of plants can be presented as the promoting growth of plants in the soil of N stress.In the present invention
One embodiment in, the soil of the N stress is specially that Northeast Area of China is (specific as inthe Shanmen area, Siping, Jilin province city Lishu County is faced
Soil Hai Zhen).
Further, the N stress is 60% that amount of application of nitrogen fertilizer is conventional fertilizer application amount, and phosphate fertilizer and potash fertilizer are constant.Institute
State conventional fertilizer application amount are as follows: N:P (P2O5): K (K2O)=9.6Kgha-1: 16Kgha-1: 12Kgha-1.In the present invention,
The nitrogenous fertilizer is specially urea.
In the present invention, the plant can be cereal crops, concretely peanut, such as white sand 308.
The present invention is directed to the Northeast's peanut production and soil status, measures the high-efficiency nitrogen-fixing peanut separated from the Northeast
The abilities such as the environmental suitability of rhizobium go out slow raw rhizobium (Bradyrhizobium sp.) from peanut nodule isolation and selection
DBPB1, the bacterial strain are adapted to the Northeast, have growth-promoting characteristic, and, tolerance pesticide for example drought-resistant with very strong adaptability
It is bio-safety bacterial strain with acid and alkali-resistance etc..In the case where reducing conventional amount of application of nitrogen fertilizer 40%, the microbial inoculum ratio is applied in field
It does not apply and compares the characters such as the peanut plant height that can dramatically increase different development stage, root nodule number, ground fresh weight, peanut yield ratio
The processing of conventional fertilizer application improves 11.2%.
The present invention provides high-efficiency nitrogen-fixing for one of main growing area of Chinese Peanut-the Northeast and stablizes the micro- of growth-promoting
Germplasm resources may advantageously facilitate the green and healthy sustainable development of Chinese agriculture.Utilize slow raw rhizobium
(Bradyrhizobium sp.) DBPB1 bacterial strain can reduce the investment of nitrogenous fertilizer and mitigate excessive applied nitrogen caused by environment
Pollution meets needs and low-carbon, environmental protection, the requirement of the ecological agriculture of the sustainable green agriculture development of China.The present invention expands
Grain in China promoting crop growth bacterium germ plasm resource provides basis to research and develop microbial manure and the application technology of efficient stable.
Biomaterial information
Strain name: slow raw rhizobium DBPB1
Latin literary fame: Bradyrhizobium sp.DBPB1
Classification naming: slow raw rhizobium Bradyrhizobium sp.
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism abbreviation: CGMCC
Deposit number: CGMCC NO.17502
Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Detailed description of the invention
Fig. 1 is colonial morphology of the bacterial strain DBPB1 on YMA solid medium.
Fig. 2 is the Gram's staining result figure of bacterial strain DBPB1.
Fig. 3 is acid solid culture medium bacterium colony figure of the bacterial strain DBPB1 in pH6.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Used in following embodiments
Experimental method unless otherwise specified, be conventional method.The materials, reagents and the like used in the following examples, such as without special theory
It is bright, it is commercially available.
The culture medium used used in following embodiments and preparation method thereof is as follows:
TY culture medium (TY solid medium and TY fluid nutrient medium)
Tryptone 5.0g, yeast powder 3.0g, CaCl20.6g, deionized water 1L, pH 6.8-7.2；121 DEG C of sterilizings
20min.Agar 15g-20g (solid medium adds).
YMA medium (YMA solid medium and YMA fluid nutrient medium)
Mannitol 10.0g, K2HPO40.25g, KH2PO40.25g, MgSO40.1g, NaCl 0.1g, yeast powder 3.0g,
Deionized water 1L, pH 6.8-7.0,121 DEG C of sterilizing 20min；Agar 15g-20g (solid medium adds).
Blood agar culture-medium
Peptone 18g, yeast powder 1g, NaCl5g, agar 15g-20g, deionized water 1L, pH6.8-7.2.121 DEG C of sterilizings
After 20min, 50 DEG C of 5% (5ml/100ml) of addition are cooled to culture medium and take off fiber sheep blood, inverted plate after mixing.
Saliferous solid medium
TY solid medium is configured, 3 parts of TY solid mediums of Xiang Wenre are separately added into the NaCl of 1g, 4g and 8g, then
Inverted plate obtains the saliferous solid medium that salinity is 1g/100ml, 4g/100ml and 8g/100ml；121 DEG C of sterilizings
20min。
Soda acid culture medium
TY solid medium is configured, the 5 parts of TY solid cultures warmed after adjusting sterilizing with sterile 0.1M HCl and NaOH
Base pH is respectively to 5.0,6.0,7.0,8.0,9.0, and then inverted plate, obtains the soda acid solid of pH5.0,6.0,7.0,8.0,9.0
(wherein, pH5.0,6.0 are acid solid culture medium to culture medium, and pH 7.0 is neutral solid medium, and pH8.0,9.0 alkalinity are solid
Body culture medium)；121 DEG C of sterilizing 20min.
TY fluid nutrient medium is configured, the 5 parts of TY Liquid Cultures warmed after adjusting sterilizing with sterile 0.1M HCl and NaOH
Then base pH is sub-packed in sterile test tube respectively to 3.0,4.0,7.0,10.0,11.0, obtain pH 3.0,4.0,7.0,
10.0, (wherein, pH3.0,4.0 are acidic liquid culture medium to 11.0 soda acid fluid nutrient medium, and pH 7.0 is neutral liquid training
Support base, pH10.0,11.0 akaline liquid culture mediums)；121 DEG C of sterilizing 20min.
Arid culture medium
YMA fluid nutrient medium is configured, 4 parts of YMA mediums of Xiang Wenre are separately added into the PEG of 0g, 10g, 20g and 30g
6000, so that the concentration (unit g/100ml) of PEG 6000 is respectively as follows: 0,10,20 and 30, it is dense to obtain four kinds of PEG 6000
Degree is respectively 0g/100ml (positive control), 10g/100ml (mild drought), 20g/100ml (mild drought) and 30g/
The arid culture medium of 100ml (Severe drought), the corresponding flow of water are respectively as follows: 0, and -0.185, -0.559, -1.122MPa；121
DEG C sterilizing 20min.
Pesticide culture medium
Peanut Common Herbicides, fungicide, insecticide are chosen, are calculated according to the preparation dosage on pesticide specification
The maximum dilution multiple of every kind of pesticide in practical applications and minimum extension rate determine suitable dilution ladder in this section
Degree, into the TY culture medium of the warm after sterilizing, be rapidly injected the pesticide of different volumes and be uniformly mixed, inverted plate, obtain with
Prepare the pesticidal solid culture medium of corresponding dilution gradient.
The concentration of pesticide used in table 1 and corresponding pesticide concentration
The functional analysis of embodiment 1, Bradyrhizobium sp Arachis (Bradyrhizobium sp.) DBPB1
One, the separation of bacterial strain DBPB1
The present invention acquires peanut plant from the Northeast, and bacterial strain is isolated and purified from the root nodule of peanut plant, identifies, obtains
To bacterial strain DBPB1.
By bacterial strain DBPB1 culture in YMA solid medium, colonial morphology: bacteria colony white, round, surface is smooth impermeable
Bright (Fig. 1), Gram's staining are feminine gender, and thallus is rod-shaped, no gemma (Fig. 2).
Two, bacterial strain DBPB1 is analyzed
1, safety detection
Bacterial strain DBPB1 is inoculated in blood agar plate culture medium, 37 DEG C of culture 7d, whether there is or not haemolysis circles for observation.There is haemolysis
It irises out and bacterial strain is represented to have hemolytic activity, have potential threat to people and animals, then bacterial strain should be forbidden being applied to microbial manure；If nothing
Haemolysis, which is irised out, represents bacterial strain without hemolytic activity, is safe strain, can be used as microbial manure application strain.
Bacterial strain DBPB1 does not occur haemolysis circle on blood agar plate, and hemolytic activity is feminine gender.
2, resistance detects
1) salt tolerant detects
By OD600It is 1g/100ml, 4g/100ml that the bacterial strain DBPB1 bacterium solution of value about 0.3 is inoculated in above-mentioned salinity respectively
On the saliferous solid medium of 8g/100ml, inoculum concentration is 10 μ L, and each processing in triplicate, is placed in 28 DEG C of insulating box trainings
It supports, observes and records the growing state of the bacterial strain DBPB1 on each concentration gradient saliferous solid medium.
The result shows that bacterial strain DBPB1 can salinity be 1g/100ml, the saliferous solid culture basal growth of 4g/100ml,
It is not grown on the saliferous solid medium that salinity is 8g/100ml, illustrates that the resistance to salinity of bacterial strain DBPB1 highest is 4g/
100ml (as shown in table 2) illustrates that the salt resistance ability of bacterial strain DBPB1 is very strong.
2) acid and alkali-resistance detects
By OD600The bacterial strain DBPB1 bacterium solution of value about 0.3 is inoculated in the acid of above-mentioned pH5.0,6.0,7.0,8.0,9.0 respectively
On alkali solid medium, wherein the culture medium of pH7.0 is positive controls, and inoculum concentration is 10 μ L, each processing in triplicate,
28 DEG C of insulating box cultures are placed in, when the bacterial strain DBPB1 growing state of positive controls is good, observe and record each gradient soda acid
The growing state of bacterial strain DBPB1 on solid medium.
By OD600The soda acid liquid training of the access of bacterial strain DBPB1 bacterium solution pH3.0,4.0,7.0,10.0,11.0 of value about 0.6
It supports in base, wherein the culture medium of pH7.0 is positive controls, and inoculum concentration is 150 μ L (3%v/v inoculum concentration), each processing weight
Multiple 28 DEG C, 180r/min shaking table culture 7d, then mixing samples three times, is measured in each soda acid fluid nutrient medium at 600nm
The OD value of bacterial strain DBPB1, with the growth and breeding situation of the size evaluation bacterial strain DBPB1 of OD value.
The result shows that bacterial strain DBPB1 can well grow (pH6 acid solid training on the soda acid solid medium of pH6-9
It is as shown in Figure 3 to support base bacterial strain DBPB1 growing state), bacterium colony growth is similar with the bacterium colony of positive controls, in the acid of pH 3-5
(i.e. OD is not grown on property fluid nutrient medium and acid solid culture medium600=0), in the neutral solution of pH 7.0,10.0 and 11.0
Bacterium solution absorbance (the OD grown on body culture medium and akaline liquid culture medium600) value is similar, it is 1.75,1.35 and respectively
1.75, illustrate that bacterial strain DBPB1 can well be grown in pH6-11 range, there is certain acid resistance and stronger alkali resistance (such as
Table 2).
3) drought-resistant detection
Flow of water manual simulation drought condition is adjusted using polyethylene glycol (PEG 6000) and carries out drought-resistant bacterial strain identification.It will
OD600The bacterial strain DBPB1 of value about 0.6 is inoculated in 0g/100ml (positive control), 10g/100ml (mild drought), 20g/ respectively
It is each to handle in triplicate in the arid culture medium of 100ml (mild drought) and 30g/100ml (Severe drought), 28 DEG C,
180r/min shaking table culture 7d measures the absorbance (OD of bacterium solution600), mean absorbance values are similar, are 5.0,5.2,4.5 respectively
With 4.3.Bacterial strain DBPB1 can be grown on the culture medium of three kinds of Middle altitude mountains, and similar with positive control, illustrate bacterial strain DBPB1
It is resistant to Severe drought, that is, the concentration for being resistant to PEG 6000 is 30g/100ml (such as table 2).
The resistance qualification result of 2 bacterial strain DBPB1 of table
Note :+indicate strain growth.
To sum up, it is 4g/100mlNaCl that the resistance of bacterial strain DBPB1, which is resistance to salinity,；Acid resistance is pH6；Alkali resistance is
pH11；Drought-resistant concentration is 30g/100mlPEG 6000, Severe drought.
3, Resistance detection
By OD600The bacterial strain DBPB1 bacterium solution of value about 0.6 is connected to respectively in the culture dish equipped with pesticide culture medium of table 1, often
A processing in triplicate, is cultivated in 28 DEG C of constant incubators, and bacterial strain DBPB1 growing state is observed after 7-10d.As a result such as 3 institute of table
Show, bacterial strain DBPB1 cannot be grown on the culture medium containing herbicide, can be grown, contained on the culture medium containing imidacloprid
It can grow on the culture medium of middle low concentration Fluoxastrobin, but cannot be grown on the culture medium containing maximum concentration.
The drug resistance qualification result of 3 bacterial strain DBPB1 of table
Note :+indicate strain growth；Indicate that bacterial strain is not grown.
To sum up, the drug resistance of bacterial strain DBPB1 is the phonetic bacterium of fungicide generally used in tolerable agricultural application as the result is shown
The maximum of ester (250mg/L) and pesticide imidacloprid (58.3mg/L) uses concentration.
4,16s rDNA sequence is identified
Utilize TreliefTMPlant Genomic DNA kit (TsingKe) kit extracts the DNA of bacterial strain DBPB1,
And using forward primer 16s rDNA P1 and reverse primer 16s rDNA P6 (sequence 2 and 3 in table 4 and sequence table) to 16s
RDNA sequence carries out PCR amplification, obtains pcr amplification product；Wherein, amplification reaction condition are as follows: 95 DEG C of 5min；94 DEG C of 1min, 60
DEG C 30s, 72 DEG C of 90s carry out 30 circulations；Last 72 DEG C extend 10min eventually.It send after the detection of PCR amplified production is qualified to Beijing
Mei Jisangge biological medicine Science and Technology Ltd. is sequenced.
4 primer information of table
Both-end sequencing sequence is spliced with DNAMAN software, and the 16s rDNA sequence of 1262bp, nucleotide is obtained
Sequence is as shown in SEQ ID No.1, and obtained 16s rDNA sequence carries out BLAST comparison in the website NCBI, as the result is shown the bacterium
Strain and Bradyrhizobiumottawaense OO99 similitude highest, homology 100%.
In view of to bacterial strain DBPB1 colonial morphology and sequencing qualification result, determining that bacterial strain DBPB1 is slow raw rhizobium above
(Bradyrhizobium sp.).Bacterial strain DBPB1 is preserved in Chinese microorganism strain preservation management on March 29th, 2019
Committee's common micro-organisms center, deposit number are CGMCC No.17502.Preservation address is Chaoyang District, Beijing City North Star west
The institute 3 of road 1.
The Field information research of embodiment 2, Bradyrhizobium sp Arachis (Bradyrhizobium sp.) DBPB1
One, the Field information method of raw rhizobium (Bradyrhizobium sp.) DBPB1 slowly
1, the slow raw rhizobium (Bradyrhizobium of -80 DEG C of refrigerators will bacterial strain activation and expansion culture: be preserved in
Sp.) DBPB1 strain is activated using hatched manner in YMA solid medium, is put in 28 DEG C of constant incubator cultures, and separation is single
Bacterium colony.Single colonie is chosen with aseptic inoculation ring into YMA fluid nutrient medium and expands culture, is put in 28 DEG C, 180 revs/min of shaking table
Shaken cultivation 7 days or so to bacterial concentration be 1 × 1010-11Cfu/ml or so (OD600=1.2 or so) microbial inoculum mother liquor, is obtained；
2, add clean tap water to be diluted to three times volume in microbial inoculum mother liquor to obtain bacterium solution and mix using preceding, then again to bacterium
Liquid microelement is added dropwise in liquid, and (amount of the liquid microelement added into the dilution bacterium solution is that every 3mL bacterium solution adds 1 μ
Liquid microelement described in L) and mix, obtain slow raw rhizobium (Bradyrhizobium sp.) DBPB1 liquid bacterial agent.
Wherein, the content of the liquid microelement each component is as follows: H3BO32.86g/L MnSO41.81g/L CuSO4·
5H2O 0.80g/L, ZnSO40.22g/L, H2MoO40.02g/L sequentially adds the above component after all dissolving in suitable quantity of water,
1L is supplied with deionized water.
3, packet microbial inoculum: peanut species uniformly will be sprayed on by raw rhizobium (Bradyrhizobium sp.) DBPB1 liquid bacterial agent slowly
Sublist face is wet to the surface of the seed, is placed on shady place and dries in the shade；
4, packet protective agent: the seed to dry in the shade the carboxymethylcellulose sodium solution (carboxymethyl cellulose of final concentration of 10g/L
Plain sodium water solution, preparation method: 10g sodium carboxymethylcellulose (800~1200mPas) is dissolved in lL deionized water, at 60 DEG C
Stirred in water bath is dissolved to transparent paste glue) seed dressing and coating (25ml/kg), microbial inoculum attachment and humidity are kept, is dried in the shade；
5, artificial or machine sowing: humidity conjunction is begun sowing in good time after broadcasting preceding moisture creating watering or rain, guarantees the necessary humidity of bacterial strain
Conducive to existence.
Two, raw rhizobium (Bradyrhizobium sp.) DBPB1 Field information example slowly
Test site is located at inthe Shanmen area, Siping, Jilin province city Lishu County and borders on the sea town；Selected peanut varieties are the local product generally used
Kind white sand 308；Sowing date is on May 23rd, 2018, broadcasts preceding watering；Experimental plot is divided into two areas: normal fertilization area is (with normal
Advise dose nitrogen fertilizer application, phosphate fertilizer and potash fertilizer) and the area Jian Dan (subtract nitrogen 40% but phosphate fertilizer and potash fertilizer are applied with conventional fertilizer application amount), it is each small
Area's area is 60 square metres, and each cell is in triplicate.Nitrogen fertilizer application is urea (active constituent content 46%N), and phosphate fertilizer is phosphorus
Sour diammonium (active constituent content 18%N+46%P2O5), potash fertilizer is potassium sulfate (active constituent content 50%K2O)；It is conventional
Dose are as follows: N:P (P2O5): K (K2O)=9.6Kgha-1: 16Kgha-1: 12Kgha-1, base manure is used as after fertilizer mixing
It is disposably manured into soil, specific dosage of applying fertilizer is as shown in table 5；Sow the peanut seed conduct without processing in normal fertilization area
Positive controls (CK), slow raw rhizobium (Bradyrhizobium sp.) the DBPB1 processing that the area Jian Dan sowing step 1 obtains
Peanut seed afterwards observes peanut full-bloom stage biomass and harvesting peanut phase biomass as processing group.
Peanut full-bloom stage biomass is as shown in table 6, and processing group and positive controls (CK) compare, the plant strain of peanut full-bloom stage
High and overground part fresh weight significantly improves, total knit stitch number (including the summation of the knit stitch number of fruit number and not formed fruit) and fruit
Real number is also improved.
Harvesting peanut phase biomass is as shown in table 7, and processing group is significantly improved than the root nodule number of positive controls (CK), strain
High and fruit number increases.The peanut fruit per mu yield 463kg of processing group, than positive controls (CK) volume increase about 11.3%.
The every cell fertilizer amount table in the normal fertilization area of table 5 and the area Jian Dan
Note: subtract phosphate fertilizer in nitrogen processing in practical fertilizer amount and decrease 8%.
6 peanut full-bloom stage biomass of table (after planting 60 days)
Note: different lowercases indicate aobvious in 0.05 horizontal upper difference
7 harvesting peanut phase of table biomass (after planting 120 days)
Note: different lowercases are indicated in 0.05 horizontal upper significant difference
Field experiment result obtains slow raw rhizobium (Bradyrhizobium sp.) DBPB1 in peanut difference growth and development
The biomass of peanut plant, root nodule number and total output can be improved in stage, which is higher than positive controls, table
This bright slow raw rhizobium (Bradyrhizobium sp.) DBPB1 can be used as the microorganism manure strain money of exploitation stability and high efficiency
Source is suitable for the similar area of environmental condition.Area as may also being not limited to environmental classes.
The present invention is had been described in detail above.To those skilled in the art, do not depart from spirit of the invention and
Range, and without carrying out under unnecessary experimental conditions, can synchronization parameters, concentration and under the conditions of, it is real in a wider range
Apply the present invention.Although The present invention gives particular embodiments, it is understood that, the present invention can be improved further.
In short, pressing the principle of the present invention, the application is intended to include any change, purposes or improvement of the present invention, including departing from this Shen
Please in the open scope, and the change carried out with routine techniques known in the art.By the model of following attached claims
It encloses, the application of some essential characteristics can be carried out.
Sequence table
<110>China Agricultural University
The slow raw rhizobium of the degeneration-resistant fixed nitrogen of<120>one plants of suitable the Northeasts and its application
<130> GNCFY191573
<160> 3
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1262
<212> DNA
<213>raw rhizobium (Bradyrhizobium sp.) slowly
<400> 2
gggaacatac cttttggttc ggaacaacac agggaaactt gtgctaatac cggataagcc 60
cttacgggga aagatttatc gccgaaagat tggcccgcgt ctgattagct agttggtgag 120
gtaatggctc accaaggcga cgatcagtag ctggtctgag aggatgatca gccacattgg 180
gactgagaca cggcccaaac tcctacggga ggcagcagtg gggaatattg gacaatgggg 240
gcaaccctga tccagccatg ccgcgtgagt gatgaaggcc ctagggttgt aaagctcttt 300
tgtgcgggaa gataatgacg gtaccgcaag aataagcccc ggctaacttc gtgccagcag 360
ccgcggtaat acgaaggggg ctagcgttgc tcggaatcac tgggcgtaaa gggtgcgtag 420
gcgggtcttt aagtcagggg tgaaatcctg gagctcaact ccagaactgc ctttgatact 480
gaagatcttg agttcgggag aggtgagtgg aactgcgagt gtagaggtga aattcgtaga 540
tattcgcaag aacaccagtg gcgaaggcgg ctcactggcc cgatactgac gctgaggcac 600
gaaagcgtgg ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgaat 660
gccagccgtt agtgggttta ctcactagtg gcgcagctaa cgctttaagc attccgcctg 720
gggagtacgg tcgcaagatt aaaactcaaa ggaattgacg ggggcccgca caagcggtgg 780
agcatgtggt ttaattcgac gcaacgcgca gaaccttacc agcccttgac atgtccagga 840
ccggtcgcag agatgtgacc ttctcttcgg agcctggaac acaggtgctg catggctgtc 900
gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cccgtcctta 960
gttgctacca tttagttgag cactctaagg agactgccgg tgataagccg cgaggaaggt 1020
ggggatgacg tcaagtcctc atggccctta cgggctgggc tacacacgtg ctacaatggc 1080
ggtgacaatg ggatgctaag gggcgaccct tcgcaaatct caaaaagccg tctcagttcg 1140
gattgggctc tgcaactcga gcccatgaag ttggaatcgc tagtaatcgt ggatcagcac 1200
gccacggtga atacgttccc gggccttgta cacaccgccc gtcacaccat gggagttggt 1260
tt 1262
<210> 2
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
agagtttgat cctggctcag aacgaacgct 30
<210> 4
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tacggctacc ttgttacgac ttcacccc 28
Claims (10)
1. one plant of slow raw rhizobium, it is characterised in that: the slow raw rhizobium are slow raw rhizobium (Bradyrhizobium
Sp.) DBPB1 is CGMCC in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center
No.17502。
2. a kind of microbial inoculum, it is characterised in that: the microbial inoculum includes slow raw rhizobium described in claim 1.
3. microbial inoculum according to claim 2, it is characterised in that: the microbial inoculum has following at least one characteristics:
A1) salt tolerant；
A2) acidproof；
A3) alkaline-resisting；
A4) drought-resistant；
A5) drug resistance；
A6) fixed nitrogen；
A7) promoting growth of plants.
4. slow raw rhizobium described in claim 1 or microbial inoculum as claimed in claim 2 have following at least one special in preparation
Application in the product of property:
A1) salt tolerant；
A2) acidproof；
A3) alkaline-resisting；
A4) drought-resistant；
A5) drug resistance；
A6) fixed nitrogen；
A7) promoting growth of plants.
5. application according to claim 4, it is characterised in that: the product is microbial manure.
6. a kind of application method of microbial inoculum, which is characterized in that the application method of the microbial inoculum includes the following steps:
S1 the bacterium solution of slow raw rhizobium described in claim 1) is added into microelement；
S2 the step S1) bacterium solution for obtaining addition microelement) is sprayed into vegetable seeds surface, is dried in the shade；
S3 seed dressing and coating) is carried out to the seed that step S2) dries in the shade with carboxymethylcellulose sodium solution, is dried in the shade；
S4 it) sows.
7. application method according to claim 6, it is characterised in that: step S1) in, the microelement is with microelement
The form of liquid is added in bacterium solution, the content of each component of the liquid microelement are as follows: H3BO32.86g/L MnSO4 1.81g/
L, CuSO4·5H2O 0.80g/L, ZnSO40.22g/L, H2MoO40.02g/L, solvent are water；
And/or the carboxymethylcellulose sodium solution is the sodium carboxymethyl cellulose solution of final concentration of 10g/L.
8. application of the application method described in claim 6 or 7 in promoting growth of plants.
9. according to application described in claim 4 or 5 or 8, or, microbial inoculum described in claim 2 or 3, it is characterised in that: described
Promoting growth of plants is presented as following at least one:
B1) plant height of the plant is promoted to increase in the different development stage of the plant；
B2) the fruit number of the plant is promoted to increase in the different development stage of the plant；
B3 the root nodule number of the plant) is promoted to increase；
B4 the knit stitch number of the plant) is promoted to increase；
B5 the ground fresh weight of the plant) is promoted to increase；
B6 the yield of the plant) is promoted to increase；
The different development stage is full-bloom stage and/or mature harvest time.
10. according to application described in claim 4 or 5 or 8, or, microbial inoculum described in claim 2 or 3, or, claim 6 or
Application method described in 7, it is characterised in that: the plant is cereal crops.
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| CN115197879A (en) * | 2022-06-29 | 2022-10-18 | 华南农业大学 | Slow rhizobium W052 and application thereof |
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| CN105567615A (en) * | 2016-03-14 | 2016-05-11 | 中国科学院烟台海岸带研究所 | Bradyrhizobium sp. and application thereof |
| CN107624577A (en) * | 2017-09-26 | 2018-01-26 | 安徽徽大农业有限公司 | One cultivates peanut the green implantation methods of high yield |
| CN109679884A (en) * | 2019-02-28 | 2019-04-26 | 中国农业大学 | One plant of efficient Promoting bacteria of corn that can be reduced nitrogen phosphorus fertilizer application and its application |
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| CN107624577A (en) * | 2017-09-26 | 2018-01-26 | 安徽徽大农业有限公司 | One cultivates peanut the green implantation methods of high yield |
| CN109679884A (en) * | 2019-02-28 | 2019-04-26 | 中国农业大学 | One plant of efficient Promoting bacteria of corn that can be reduced nitrogen phosphorus fertilizer application and its application |
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