CN107828701A - A kind of take-all biocontrol bacterial strain, its microbial inoculum and application - Google Patents
A kind of take-all biocontrol bacterial strain, its microbial inoculum and application Download PDFInfo
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- CN107828701A CN107828701A CN201711340409.8A CN201711340409A CN107828701A CN 107828701 A CN107828701 A CN 107828701A CN 201711340409 A CN201711340409 A CN 201711340409A CN 107828701 A CN107828701 A CN 107828701A
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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
- C12R2001/38—Pseudomonas
- C12R2001/39—Pseudomonas fluorescens
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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
Abstract
The present invention relates to take-all biocontrol bacterial strain, its microbial inoculum and application.The bacterial strain is Pseudomonas fluorescens (Pseudomonas fluorescens) GP154, and preserving number is CGMCC No.14667.The microbial inoculum contains foregoing bacterial strain.The purposes of the microbial inoculum is preventing and treating take-all.Microbial inoculum of the present invention has obvious prevention effect to take-all, compensate for the not good enough deficiency of chemical agent preventive effect, and a kind of means are added for the integrated control of take-all；Microbial inoculum preparation technology is simple, is adapted to industrialized production.
Description
Technical field
The present invention relates to a kind of take-all biocontrol bacterial strain, its microbial inoculum and application, belongs to agriculture biocontrol microorganisms technical field.
Background technology
According to the inventors knowledge, take-all (Take-all), also known as " black pin " disease, it is a kind of typical root soil-borne disease
Evil, is mainly caused by Gaeumannomyces graminis var.avenae Gaeumannomyces graminis var.tritici (Ggt).Pathogen
The general position for infecting wheat root and basal part of stem 1-2 sections, take-all can occur in seedling stage and maturity period, jointing stage disease
Strain symptom is more obvious, and blade turns yellow, and old complaint blackening, when wheat comes to the ripening period, dead ears symptom occurs.
Take-all is found most earlier than 1852 in South Australia, later in North of Italy Bermuda grass and wheatgrass
Take-all is found on dead stem, is occurred in succession in every country afterwards, and is on the rise, the yield of wheat is caused huge
Loss.1931, take-all found that take-all is in China Jiangsu, Anhui, river at present in Zhejiang Province of China for the first time
There are different degrees of generation in 22 provinces such as south, wherein the most serious, the 2012 years onset areas in Henan occur with Henan, Shandong and other places
4500000 mu.Full rot destroys wheat root, can cause the aggrieved wheatland underproduction 20%~50%, and severe patient is even had no harvest.Full rot
It has been listed in the supplement quarantine disease of multiple provinces such as Henan, Jiangsu, Anhui.
Although existing researcher proposes complete come integrated control wheat using the means such as agricultural measures and seed selection disease-resistant variety
Erosion disease, but including crop rotation, increase the agricultural measures such as acid-base value of fertilizer and regulation soil because operability is not strong and seldom quilt
Using；The seed selection of take-all resistant variety is also in the exploratory stage.Preventing and treating at present in production to take-all is main
Or carry out seed treatment by chemical bactericide.The bactericide commonly used for take-all has Silthiopham, phenyl ether methyl cyclic
Azoles and Tebuconazole etc., because take-all occurs in root, these medicaments can only do Dressing.These chemicals treatments are small
The early stage of wheat growth can show good effect, but to the middle and later periods, and their control effects to take-all are but very not
Ideal, this is caused by medicament decay in the environment.In addition to the lasting period is short, the use of these chemical agents can also give environment
Certain negative effect is caused, therefore, is solely just a makeshift arrangement by chemical bactericide to prevent and treat take-all.
Take-all has dieback phenomenon, i.e., in continuous cropping wheat paddock, after full rot peak is developed into, is not taking
In the case of any prophylactico-therapeutic measures, it may appear that the phenomenon that full rot mitigates or even disappeared naturally.Substantial amounts of research shows both at home and abroad
The decline of take-all mainly due to caused by the antagonistic microbe in soil, this prompting researcher using drip irrigation come
It is perhaps exactly a good selection to control take-all.
As the project team of research wheat diseases, inventor has carried out the biological control research work of take-all, sieve
Choosing has the bacterial strain of notable antagonism to gaeumannomyces graminis, and is made into microbial inoculum, does not have commercialization also at home and abroad
Take-all biocontrol agent background under, this work has great importance.
Before, inventor by existing achievement in research application and obtains 1 Chinese invention patent：Patent No.
CN201310630644.4, Authorization Notice No. CN103602621B, it is CGMCC to be directed to a kind of preserving number：8431 it is green
It pin pseudomonad, can be used to prevent and treat take-all, there is obvious prevention effect, it is not good enough not to compensate for chemical agent preventive effect
Foot；Because Pseudomonas chlororaphis bacterial strain can colonize in wheat rhizosphere, so the microbial inoculum has the longer lasting period；And it is not using
Environmental problem can be brought, is advantageous to the No-harmful apple orchard of wheat.Hereafter, inventor continues to be directed to research and development preventing and treating take-all
Biocontrol bacterial strain and microbial inoculum.
The content of the invention
Newest research results of the invention based on inventor, there is provided a kind of take-all biocontrol bacterial strain, can be in wheat
Rhizosphere colonization, there is antagonism to gaeumannomyces graminis；Also provide and use biocontrol agent made of the bacterial strain, realize to wheat
The prevention and control of full rot.
The technical scheme that the present invention solves its technical problem is as follows：
A kind of take-all biocontrol bacterial strain, it is Pseudomonas fluorescens (Pseudomonas fluorescens) GP154,
Preserving number is CGMCC No.14667.
The present invention also provides：Take-all biocontrol bacterial strain described previously is used to prepare preventing and treating take-all biocontrol agent
Purposes.
The present invention also provides：A kind of take-all biocontrol agent, contain previously described take-all biocontrol bacterial strain.
The present invention also provides：A kind of preparation method of take-all biocontrol agent, it is characterized in that, comprise the following steps：
The first step, by previously described take-all biocontrol bacterial strain access equipped with LB fluid nutrient mediums triangular flask in,
The concussion and cultivate in shaking table, carry out the activation of strain；Shaking table temperature is 32 DEG C ± 1 DEG C；
Second step, LB fluid nutrient mediums are fitted into seeding tank, after steam sterilizing cooling, by activated spawn obtained by the first step
Seeding tank is accessed, carries out the culture of seed liquor；Cultivation temperature is 32 DEG C ± 1 DEG C；
3rd step, fermenting culture medium is fitted into fermentation tank, after sterilizing cooling, seed liquor obtained by second step is moved into
Fermentation tank, viable count during fermentation ends in zymotic fluid are 6.5 ± 0.5 × 109Individual/ml；Fermentation temperature is 32 DEG C ± 1 DEG C；
The fermenting culture medium is made up of by weight percentage following components：
Beancake powder 1.5 ± 0.5%, sweet potato starch 1.2 ± 0.3%, fish meal 0.3 ± 0.1%, corn steep liquor 0.7 ± 0.2%,
Precipitated calcium carbonate 0.175 ± 0.05%, sodium hydroxide 0.1 ± 0.05%, defoamer 0.25 ± 0.1%, remaining is water；
4th step, by zymotic fluid centrifuging and taking sediment obtained by the 3rd step, sediment and peat are mixed and stirred for uniformly, producing small
Wheat full rot biocontrol agent；Viable count in the take-all biocontrol agent is at least 5,000,000,000/gram.
Preferably, in the first step, the take-all biocontrol bacterial strain is stored in test tube slant before access；Shaking speed
For 120 ± 10r/min, incubation time is 24 ± 4 hours.
Preferably, in second step, the fluid nutrient medium volume of loading accounts for the 60 ± 10% of seed tank volume；The seeding tank
Running parameter be：150 ± 20r/min of mixing speed, dissolved oxygen 30 ± 3%, tank press 0.05 ± 0.01MPa；Incubation time is
12 ± 2 hours.
Preferably, in the 3rd step, the fermenting culture volume of loading accounts for the 70 ± 10% of fermenter volume；Second step
Gained seed liquor is moved into fermentation tank immediately when seed liquor culture terminates；The running parameter of the fermentation tank is identical with seeding tank；
Fermentation time is 18 ± 3 hours.
Preferably, disc centrifuge is used in the 4th step, during centrifugation, centrifugal rotational speed is 7000 ± 300r/min；It is described
The fineness of peat is 200 ± 50 mesh；The weight ratio of the peat and sediment is 1:1-3；In the take-all biocontrol agent
Water content be weight percentage 30 ± 5%.
The present invention also provides：Take-all biocontrol agent described previously is used for the purposes for preventing and treating take-all.
Preferably, in use, first by take-all biocontrol agent and wheat seed by weight 1:8-12 is dressed seed,
Broadcasted sowing again.
The take-all biocontrol agent of the present invention has obvious prevention effect to take-all, compensate for chemical drugs
The deficiency that agent preventive effect is not good enough, a kind of means are added for the integrated control of take-all；Microbial inoculum preparation technology is simple, is adapted to
Industrialized production.
The take-all biocontrol bacterial strain of the present invention, i.e. Pseudomonas fluorescens (Pseudomonas fluorescens)
It GP154, can be colonized in wheat rhizosphere, make biocontrol agent that there is the longer lasting period；With prominent producing IAA ability, to small
The growth-promoting functions of wheat are notable；The bacterial strain is inherently isolated from the rhizosphere of wheat, environmentally friendly, environment friendly and pollution-free, beneficial to wheat
No-harmful apple orchard.
Embodiment
Below by embodiment, the present invention will be further described, but the present invention is not limited only to these examples.The present invention
Involved reagent is purchased in market.The experimental method of following embodiments, it is conventional method unless otherwise instructed.
The separation screening of embodiment 1, potentiality bacterial strain
(1) separate
The soil sample 10g adsorbed on wheat root is taken, adds in 90ml sterilized waters, fully shaking, obtains 10-1Dilution；Continue
Sterilized water dilution is added, obtains 10-5、10-6With 10-7Dilution, take the above-mentioned three kinds of dilutions of 0.2ml respectively on LB flat boards
Coating, 25 DEG C are cultivated 48 hours, and picking single bacterium colony is inoculated into LB culture medium slants and preserved；1051 plants of bacteriums are isolated,
They are numbered.
(2) screen
A), 1051 plants of bacteriums that step (1) separates are inoculated into 1ml LB fluid nutrient mediums respectively, in 28 DEG C of 120r/
Concussion and cultivate 12 hours under min；
B), by Gaeumannomyces graminis var.avenae (Gaeumannomyces graminis var.tritici) High pathogenicity bacterium
Strain G1037 (hereinafter referred to as G1037) 5mm bacterium dish is inoculated into the center of PDA plate, after cultivating 48 hours, in G1037 bacterium colonies
Surrounding connects bacterium in step A in LB fluid nutrient mediums along the equidistant position of four direction with the toothpick point of sterilizing, and 72 is small
When after measure antibacterial bandwidth of every kind of bacterium to G1037, just sift out 25 plants have antagonism bacterial strains, then again with identical
Method carry out secondary screening.Pick out 9 plants of bacteriums with obvious antibacterial band, their numbering be respectively No.3, No.16,
No.20、No.38、No.97、No.147、No.154、No.155、No.188。
The preparation of embodiment 2, potentiality bacterial strain bacteria suspension
The 9 plants of bacteriums and GP51 bacterial strains that embodiment 1 is sieved are inoculated into the LB culture mediums in triangular flask respectively,
(condition of culture especially cultivation temperature, is decided by Optimal Experimental to concussion and cultivate within 24 hours under 32 DEG C of 120r/min
, detailed process does not repeat herein), make viable count ＞ 2.0 × 10 in the nutrient solution of each bacterial strain9cfu/ml.Each bacterial strain takes in right amount
Nutrient solution, it is diluted to 1.0 × 109Cfu/ml bacteria suspension, it is standby.
Wherein, (Patent No. CN201310630644.4, is awarded the Chinese invention patent before GP51 bacterial strains are inventor
Power notification number is CN103602621B) in Pseudomonas chlororaphis CGMCC：8431.
Embodiment 3, potentiality bacterial strain are to Wheat Seedling full rot control effect
Computational methods involved by the present embodiment：
(1) disease index
First the incidence of wheat root is classified, further according to the level calculation disease index of the state of an illness.
Severity Scaling
0 grade：It is disease-free
1 grade：Root system onset area accounts for 1%~5%；
3 grades：Root system onset area accounts for 6%~20%；
5 grades：Root system onset area accounts for 21%~40%；
7 grades：Root system onset area accounts for 41%~60%；
9 grades：Root system onset area accounts for more than 61%.
With each bacterial strain bacteria suspension of the gained of embodiment 2 (quality of wheat seed and microbial inoculum of soaking wheat seed respectively 5 minutes
Volume ratio is 1:10), then wheat seed is pulled out and dried.
The mixture of soil and river sand is filled in Pen Portland, G1037 a diameter of 5mm bacterium dish is connect on its surface, then covers 1 again
The mixture of soil and sand of cm thick.The wheat seed treated through different bacterium bacteria suspension is sowed in Pen Portland, while blank is set
Control and chemicals treatment control.Blank control：Wheat seed is soaked 5 minutes in clear water, is sowed at after drying in Pen Portland.Medicament
Processing control：With 30 g/l of suspension seed-coating agents of Difenoconazole routinely dosage (pesticide-seeds ratio 1:200) wheat seed is carried out
Dressing, it is sowed at after drying in Pen Portland.Each processing is repeated 3 times, and the disease index and preventive effect of take-all are investigated after 30 days.
As a result it is as shown in table 1.
Table 1, each potentiality bacterial strain are to the control effect of Wheat Seedling full rot
| Processing | Emergence rate (%) | Disease index | Preventive effect (%) |
| 1 (No.3 bacterial strains) | 100 | 23.33bc | 58.55ab |
| 2 (No.16 bacterial strains) | 100 | 21.85bc | 61.18ab |
| 3 (No.20 bacterial strains) | 100 | 26.67bc | 52.63ab |
| 4 (No.38 bacterial strains) | 100 | 22.22bc | 60.53ab |
| 5 (No.97 bacterial strains) | 100 | 24.81bc | 55.92ab |
| 6 (No.147 bacterial strains) | 100 | 24.81bc | 55.92ab |
| 7 (No.154 bacterial strains) | 100 | 18.52c | 67.11a |
| 8 (No.155 bacterial strains) | 100 | 20.37c | 63.82a |
| 9 (No.188 bacterial strains) | 100 | 30.37b | 46.05b |
| 10 (GP51 bacterial strains) | 100 | 21.48bc | 61.84ab |
| 11 (Difenoconazoles) | 100 | 21.85bc | 61.18ab |
| 12 (clear water) | 100 | 56.30a | -- |
The detection of embodiment 4, potentiality bacterial strain to seedling stage wheat growth-promoting effect
(1) strain secretes IAA performance measurements
The IAA of strain secretes is quantitative determined using Salkowski colorimetric methods.
(2) test of the bacterial strain to seedling stage wheat growth-promoting functions
It will be soaked 5 minutes with each bacterial strain bacteria suspension of the gained of embodiment 2 respectively after wheat packet after vernalization, and use clear water
Compare；Afterwards, wheat is sowed in the basin alms bowl equipped with vermiculite, be placed in greenhouse cultivate, after 3 weeks distinguish measurement processing group with it is right
According to the plant height of group wheat, single-strain fresh weight and dry weight.As a result it is as shown in table 2.
Table 2, potentiality strain secretes IAA performance and the growth-promoting effect to seedling stage wheat
The determination of embodiment 5, aimed strain
From embodiment 3 and the result of embodiment 4, in all potentiality bacterial strains, No.154 bacterial strains are to take-all
Control effect it is best, and its control effect is better than GP51 bacterial strains；No.154 bacterial strains have good facilitation to wheat growth,
Its auximone IAA content highest secreted, and its growth-promoting functions to wheat is much better than GP51 bacterial strains.Managed based on more than
By it is aimed strain to determine No.154 bacterial strains.
The identification and preservation of embodiment 6, aimed strain
It is observed that the morphological feature of No.154 bacterial strains is：Gram-negative, 1.0 μm of about 0.5 μ m of shaft-like, size, no bud
Spore, single polar flagella, the bacterium colony on KMB culture mediums is circular, milky, and surface is glossy, neat in edge.
The physio-biochemical characteristics of No.154 bacterial strains such as table 3.
The physio-biochemical characteristics of table 3, No.154 bacterial strains
| Character | Performance | Character | Performance | Character | Performance |
| 4 DEG C of growths | + | Peptone utilizes | + | Inositol utilizes | _ |
| 28 DEG C of growths | + | Ammonium chloride utilizes | + | D-glucitol utilizes | _ |
| 41 DEG C of growths | _ | Potassium nitrate utilizes | + | L-Histidine utilizes | + |
| Contact enzyme reaction | + | Glucose utilization | + | L-arginine utilizes | + |
| The double hydrolysis of arginine | + | Sucrose utilizes | _ | Altheine utilizes | + |
| Starch Hydrolysis | _ | Galactose utilization | _ | Thermophilic iron element | + |
| Gelatin liquefaction | + | Trehalose utilizes | _ | Protease | + |
| Fluorchrome | + | Xylose utilization | _ | Cellulase | _ |
| Beef extract utilizes | + | Mannitol utilizes | _ | Lipopeptid | + |
The sequencing of Nanjing Jin Sirui companies will be entrusted after the 16S rDNA of No.154 bacterial strains fragment clone, the sequence measured exists
BLAST is carried out in NCBI websites.As a result show, the 16SrDNA partial sequences and Pseudomonas of No.154 bacterial strains
fluorescens strain Pf0-1、Pseudomonas fluorescens strain Mc07、Pseudomonas
Fluorescens strain FE1326 16SrDNA all or part sequence homologies are up to 100%, with reference to the bacterial strain
Morphological feature and physiological and biochemical analysis, the bacterial strain is accredited as Pseudomonas fluorescens (Pseudomonas fluorescens).
It is GP154 by the bacterial strain official number.
GP154 bacterial strains are sent into China Committee for Culture Collection of Microorganisms's common micro-organisms center (China
General Microbiological Culture Collection Center, CGMCC) preservation is carried out, its address is Beijing
The institute 3 of city Chaoyang District North Star West Road 1, Institute of Microorganism, Academia Sinica, postcode is 100101, preserving number CGMCC
No.14667。
The preparation of embodiment 7, GP154 microbial inoculums
The preparation of GP154 microbial inoculums comprises the following steps：
(1) actication of culture
The GP154 bacterial strains preserved in test tube slant access is equipped with the triangular flask of 1000ml LB fluid nutrient mediums, 32
Concussion and cultivate 24 hours, carries out the activation of strain under DEG C 120r/min.
(2) seed liquor culture
Load 60L LB fluid nutrient mediums in 100L seeding tank, will be activated after steam sterilizing cooling through step (1)
Strain accesses wherein, carries out the production of seed liquor.The running parameter of seeding tank is：Temperature：32 DEG C, mixing speed：150r/min,
Dissolved oxygen：30%, tank pressure：0.05MPa.Culture 12 hours, obtains the seed liquor of strong vigor.
(3) ferment
Load 1400L special culture medias in 2000L fermentation tank.While the seed liquor production of step (2), to hair
Culture medium is sterilized and cooled down in fermentation tank.Seeding tank immediately moves into seed liquor in fermentation tank after terminating culture, is fermented
Production, the same seeding tank of running parameter of fermentation tank, fermentation is terminated after 18 hours, in zymotic fluid GP154 viable count for 6.5 ×
109Individual/ml.
Optimized GP154 bacterial strain special culture media formulas：Beancake powder 1.5%, sweet potato starch 1.2%, fish meal 0.3%,
Corn steep liquor 0.7%, precipitated calcium carbonate 0.175%, sodium hydroxide 0.1%, defoamer (such as modified polyorganosiloxane) 0.25% are molten
Agent is water.
(4) microbial inoculum is processed
The zymotic fluid of step (3) is centrifuged through disc centrifuge, centrifuge speed 7000r/min, liquid is abandoned, obtains
Sediment.Sediment and fineness are pressed 1 for the peat of 200 mesh:1.6 (weight) mix, and are sufficiently stirred, produce microbial inoculum.Microbial inoculum water content
For 30%, wherein GP154 viable counts are more than 5,000,000,000/gram.
Wherein, zymotic fluid is concentrated with disc centrifuge, efficiency high, energy consumption is low, and is provided for lower procedure
It is convenient.
The field test of embodiment 8, GP154 microbial inoculums
Experiment is carried out in certain town take-all recurrence field, and material therefor is GP154 microbial inoculums, GP51 microbial inoculums, phenylate
Methyl cyclic-azole seed coat agent (trade name opposes the pellet that withers).GP154 microbial inoculums are the microbial inoculum prepared by embodiment 7, and GP51 microbial inoculums are trained for laboratory
Foster bacterium solution obtains precipitation after desk centrifuge centrifuges and gained is mixed with peat, and the two viable count is essentially identical.
GP154 microbial inoculums, GP51 microbial inoculums respectively with wheat seed by weight 1:Broadcast sowing that (wheat seed dosage is pressed after 10 seed dressings
10kg/ mus are converted), Difenoconazole seed coat agent is coated processing to wheat by its specification.Respectively 3 repetitions of processing, cell
Area 66.7m2, random district's groups arrangement.Jointing stage and the disease index of heading stage investigation take-all, investigation dead ears before harvesting
Rate.As a result it is as shown in table 4.
Control effect of the processing such as table 4, GP154 microbial inoculums to wheat Adult plant full rot
Result above shows, under the natural conditions of field, GP154 microbial inoculums can continuously and effectively prevention and control take-all, its
Positive effect is better than the processing of chemical agent Difenoconazole seed pelleting, is also better than GP51 microbial inoculums.GP154 microbial inoculums can be continual and steady
Ground prevention and control take-all, show that the bacterial strain can grow and breed well in wheat root, its Population will not be with the time
Extension and reduce rapidly, fully demonstrated its advantage as biocontrol microorganisms.
Although in addition, application number CN201110266283.0 application publication numbers CN102286416A Chinese invention patent Shen
Please, application number CN200510112542.9 Authorization Notice No. CN100390272C Chinese invention patent, application number
CN201410386364.8 Authorization Notice No. CN104152380B Chinese invention patent individually discloses can prevention and control wheat total eclipse
The pseudomonas fluorescens strain of disease, but from condition of culture, the strain culturing temperature of above-mentioned patent or patent application is most
Height is only 30 DEG C, and the cultivation temperature needed for GP154 bacterial strains of the present invention is 32 DEG C ± 1 DEG C, and both are fundamental differences；Meanwhile
GP154 bacterial strains of the present invention need to use special fermentation medium in fermentation, and the culture medium is that inventor is directed to GP154 of the present invention
Bacterial strain just draws through in depth repeatedly practising in research, is not directed in above-mentioned patent or patent application related to this interior
Hold.It is a kind of new pseudomonas fluorescens strain that these, which can confirm GP154 bacterial strains of the present invention, with these published fluorescence
Pseudomonad strain is different.
In addition to the implementation, the present invention can also have other embodiment.It is all to use equivalent substitution or equivalent transformation shape
Into technical scheme, all fall within the protection domains of application claims.
Claims (10)
1. a kind of take-all biocontrol bacterial strain, being Pseudomonas fluorescens (Pseudomonas fluorescens) GP154, protect
Tibetan number is CGMCC No.14667.
2. take-all biocontrol bacterial strain described in claim 1 is used for the purposes for preparing preventing and treating take-all biocontrol agent.
3. a kind of take-all biocontrol agent, contain the take-all biocontrol bacterial strain described in claim 1.
4. a kind of preparation method of take-all biocontrol agent, it is characterized in that, comprise the following steps：
The first step, by described in claim 1 take-all biocontrol bacterial strain access equipped with LB fluid nutrient mediums triangular flask in,
The concussion and cultivate in shaking table, carry out the activation of strain；Shaking table temperature is 32 DEG C ± 1 DEG C；
Second step, LB fluid nutrient mediums are fitted into seeding tank, after steam sterilizing cooling, activated spawn obtained by the first step is accessed
Seeding tank, carry out the culture of seed liquor；Cultivation temperature is 32 DEG C ± 1 DEG C；
3rd step, fermenting culture medium is fitted into fermentation tank, after sterilizing cooling, seed liquor obtained by second step is moved into fermentation
Tank, viable count during fermentation ends in zymotic fluid are 6.5 ± 0.5 × 109Individual/ml；Fermentation temperature is 32 DEG C ± 1 DEG C；
The fermenting culture medium is made up of by weight percentage following components：
Beancake powder 1.5 ± 0.5%, sweet potato starch 1.2 ± 0.3%, fish meal 0.3 ± 0.1%, corn steep liquor 0.7 ± 0.2%, lightweight
Calcium carbonate 0.175 ± 0.05%, sodium hydroxide 0.1 ± 0.05%, defoamer 0.25 ± 0.1%, remaining is water；
4th step, by zymotic fluid centrifuging and taking sediment obtained by the 3rd step, sediment and peat are mixed and stirred for uniformly, it is complete producing wheat
Lose disease biocontrol agent；Viable count in the take-all biocontrol agent is at least 5,000,000,000/gram.
5. preparation method according to claim 4, it is characterized in that, in the first step, the take-all biocontrol bacterial strain is connecing
Test tube slant is stored in before entering；Shaking speed is 120 ± 10r/min, and incubation time is 24 ± 4 hours.
6. preparation method according to claim 4, it is characterized in that, in second step, the fluid nutrient medium volume of loading accounts for seed
The 60 ± 10% of tank volume；The running parameter of the seeding tank is：150 ± 20r/min of mixing speed, dissolved oxygen 30 ± 3%, tank
Press 0.05 ± 0.01MPa；Incubation time is 12 ± 2 hours.
7. preparation method according to claim 4, it is characterized in that, in the 3rd step, the fermenting culture volume of loading accounts for
The 70 ± 10% of fermenter volume；Gained seed liquor is moved into fermentation tank immediately when the culture of second step seed liquor terminates；The hair
The running parameter of fermentation tank is identical with seeding tank；Fermentation time is 18 ± 3 hours.
8. preparation method according to claim 4, it is characterized in that, disc centrifuge is used in the 4th step, during centrifugation, is centrifuged
Rotating speed is 7000 ± 300r/min；The fineness of the peat is 200 ± 50 mesh；The weight ratio of the peat and sediment is 1:1-3；
Water content in the take-all biocontrol agent is weight percentage 30 ± 5%.
9. any one of claim 4 to the 8 take-all biocontrol agent is used for the purposes for preventing and treating take-all.
10. purposes according to claim 9, it is characterized in that, in use, first by take-all biocontrol agent and Wheat Species
Son is by weight 1:8-12 is dressed seed, then is broadcasted sowing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711340409.8A CN107828701B (en) | 2017-12-14 | 2017-12-14 | Wheat take-all biocontrol strain, microbial inoculum and application thereof |
Applications Claiming Priority (1)
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| CN109880757A (en) * | 2019-03-04 | 2019-06-14 | 西北大学 | One plant of hydrogen-oxidizing bacterium and its separation method and application with itself nitrogen fixing capacity |
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| CN102286416A (en) * | 2011-09-08 | 2011-12-21 | 南京农业大学 | Biocontrol bacterial strain for preventing and curing wheat takeall and application thereof |
| CN104152380A (en) * | 2014-08-07 | 2014-11-19 | 领先生物农业股份有限公司 | Ultraviolet mutagenesis type pseudomonas florescens and application thereof |
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| CN102286416A (en) * | 2011-09-08 | 2011-12-21 | 南京农业大学 | Biocontrol bacterial strain for preventing and curing wheat takeall and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109880757A (en) * | 2019-03-04 | 2019-06-14 | 西北大学 | One plant of hydrogen-oxidizing bacterium and its separation method and application with itself nitrogen fixing capacity |
| CN109880757B (en) * | 2019-03-04 | 2022-05-17 | 西北大学 | Hydrogen hydroxide bacterium with self nitrogen fixation capacity and separation method and application thereof |
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