CN105779342A - Antagonistic bacterial strain of special-form fusarium moniliforme of radix pseudostellariae and application of antagonistic bacterial strain - Google Patents
Antagonistic bacterial strain of special-form fusarium moniliforme of radix pseudostellariae and application of antagonistic bacterial strain Download PDFInfo
- Publication number
- CN105779342A CN105779342A CN201610181836.5A CN201610181836A CN105779342A CN 105779342 A CN105779342 A CN 105779342A CN 201610181836 A CN201610181836 A CN 201610181836A CN 105779342 A CN105779342 A CN 105779342A
- Authority
- CN
- China
- Prior art keywords
- radix pseudostellariae
- antagonistic
- fusarium moniliforme
- culture medium
- pseudomonas
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- 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
-
- 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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention provides an antagonistic bacterial strain of special-form fusarium moniliforme of radix pseudostellariae and application of the antagonistic bacterial strain. Antagonistic bacteria are identified as pseudomonas LK-313 with a preservation number of CGMCC NO.12121. The antagonistic bacteria provided by the invention is obtained by screening from rhizosphere soil of radix pseudostellariae through a pseudomonas selective culture medium; plate confront culture shows that the antagonistic bacteria can effectively restrain mycelial growth of the pathogenic bacteria such as the fusarium moniliforme of the radix pseudostellariae, is good in rhizospere competition and is free of pathogenicity on other after-culture crops. Meanwhile, the invention provides the optimal culture medium for growth of antagonistic bacteria, wherein the optimal formula comprises 1/4 LB, 1/20 MS (not containing ferric salt), and 0.1wt% of brown sugar, so that fermented liquid has obvious antagonistic effect. In order to overcome or relieve replanting disease of medicinal plant radix pseudostellariae, the invention provides antagonistic bacteria culture and a preparation, which are good in bio-control effect, free from pollution, safe and ecological, and wide in development application prospect.
Description
Technical field
The present invention relates to a strain bacterial isolates, particularly to a strain, Radix Pseudostellariae specialized form fusarium moniliforme is had inhibitory action
Antagonistic bacterium, can be used for overcoming or alleviated by continuous cropping Radix Pseudostellariae soil-borne disease problem, belong to microorganism and biological prevention neck
Territory.
Background technology
Radix Pseudostellariae (Pseudostellaria heterophylla) another name virgin ginseng, Radix Ginseng, meter Can, for Caryophyllaceae for many years
Raw herbaceous plant, is used as medicine with root, has the effects such as replenishing QI to invigorate the spleen, promoting the production of body fluid for nourishing the lung, and its medication is with a long history, clinical efficacy is true
Cut, the most determined by Ministry of Public Health and listed " can be used for the Chinese crude drug list of health food " in.Radix Pseudostellariae main product in Fujian, Guizhou,
The ground such as Anhui, wherein the longest with Zherong County, Fujian Province cultivation history, best in quality, have " township of China's Radix Pseudostellariae " beautiful
Reputation.But, there is serious Soil-sickness Problem (as shown in Figure 1) in Radix Pseudostellariae during cultivating and growing, continuous cropping Radix Pseudostellariae is planted
Strain retarded growth, underground part can not normally expand, and soil-borne disease is serious, and generally after results, same plot must be after 2 ~ 4 years
Can plant again, the sustainable production of serious restriction Radix Pseudostellariae and utilization.It is bad that Soil-sickness Problem also results in a series of downstream
The phenomenons such as problem, as Radix Pseudostellariae Genuine producing area and scale reduce the most year by year, even occurs moving outside producing region, genuineness distortion.Meanwhile,
In the case of the continuous cropping obstacle origin cause of formation and the mechanism of action are still not clear, major part medicinal herb grower attempts by enriching fertilizer, pesticide abuse
Maintain Radix Pseudostellariae yield, but effect is the best, but improve production cost, also result in environmental pollution, Chinese crude drug agriculture residual
Exceed standard and the problem such as farmland ecosystem functional deterioration, make the production of Radix Pseudostellariae be absorbed in vicious cycle.Therefore, Soil-sickness Problem
Research is the ecological important content urgently to be resolved hurrily of current resources of medicinal plant, becomes the focus of colleague's research both at home and abroad.
The Biological control utilizing useful Antagonistic Fungi to carry out plant soil-borne diseases is considered as a kind of science, reasonable, efficient, ring
The measure of border friendly.Seminar's early-stage Study finds, fusarium moniliforme (as shown in Figure 2) is to cause Radix Pseudostellariae soil-borne disease ferocious
Rampant important pathogen, its quantity is continuously increased with the increase of the Radix Pseudostellariae continuous cropping time limit.Further, this pathogen has host spy
The opposite sex, the most only infects Radix Pseudostellariae and does not infect other crops of rear stubble (such as Oryza sativa L., Semen sojae atricolor etc.), and the most only screening can be the most short of money
The Antagonistic Fungi of anti-Radix Pseudostellariae specialized form fusarium moniliforme could effectively prevent and treat the soil-borne disease of Radix Pseudostellariae.Meanwhile, Antagonistic Fungi to have
Stronger rhizospere competition is had could effectively to resist disease and protection plant, owing to there is coevolution between different biologies,
So being directly separated Antagonistic Fungi from disease position (such as continuous cropping Radix Pseudostellariae rhizosphere soil) is to obtain high colonization ability, strong antagonistic ability
The fundamental way of probiotics.The present invention is with Radix Pseudostellariae this pathogen of specialized form fusarium moniliforme as target, from continuous cropping Radix Pseudostellariae
In rhizosphere soil separation screening to a strain can the antagonistic bacterium of efficient this pathogenic fungi of antagonism, be identified as pseudomonas, life
Entitled Pseudomonas protegens LK-313(Pseudomonas protegens sp. nov., widespread
plant-protecting bacteria producing the biocontrol compounds 2,4-
diacetylphloroglucinol and pyoluteorin.Alban Ramette, Michele Frapolli,
Marion Fischer-Le Saux. et.Systematic and Applied Microbiology, 2011 (34): 180-
188.).
Summary of the invention
It is an object of the invention to provide Antagonistic Fungi and the application thereof of a plant height effect antagonism Radix Pseudostellariae specialized form fusarium moniliforme,
Soil-borne disease problem common in the continuous planting process of Biological control Radix Pseudostellariae, for overcoming or alleviated by Radix Pseudostellariae continuous cropping obstacle
Problem provides biocontrol bacterial strain.
Technical scheme is as follows:
Antagonistic bacterium provided by the present invention is pseudomonas (Pseudomonas protegens) LK-313, this bacterial strain in
The China of the Institute of Microorganism, Academia Sinica that on January 28th, 2016 is preserved in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City is micro-
Biological inoculum preservation administration committee's common micro-organisms center, deposit number is: CGMCC NO. 12121.
Pseudomonas of the present invention (Pseudomonas protegens) LK-313 is to Radix Pseudostellariae specialized pathogen
Bacterium fusarium moniliforme has the strongest antagonism.
The production medium of the Antagonistic Fungi of described antagonism Radix Pseudostellariae specialized form fusarium moniliforme, it is characterised in that: it is raw
Long culture medium is the 1/4LB culture medium+1/20 MS culture medium+0.1wt.% brown sugar without iron salt.
Described without iron salt its formula of MS culture medium is: 1) a great number of elements: 1900 mg/L KNO3、1650 mg/L
NH4NO3、370 mg/L MgSO4•7H2O、170 mg/L KH2PO4、440 mg/L CaCl2•2H2O；2) trace element: 22.3
mg/L MnSO4•4H2O、8.6 mg/L ZnSO4•7H2O、6.2 mg/L H3BO3、0.83 mg/L KI、0.25 mg/L
Na2MoO4•7H2O、0.025 mg/L CuSO4•5H2O、0.025 mg/L CoCl、2.6 mg/L H2O；3) Organic substance: 2.0
Mg/L glycine, 0.5 mg/L pyridoxine hydrochloride, 0.1 mg/L Tyiamine Hd element, 0.5 mg/L nicotinic acid, 100 mg/L fleshes
Acid；Described LB culture medium is: tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L.
Useful Antagonistic Fungi of the present invention has several advantages that
(1) Antagonistic Fungi pseudomonas of the present invention (Pseudomonas protegens) LK-313 is by substantial amounts of
Screening operation screens, and screens and obtains, have Radix Pseudostellariae specialized form fusarium moniliforme from 1600 strain soil bacterias
The strongest antagonistic effect, and there is cellulose degradation ability (as shown in Figure 3,4).
(2) Antagonistic Fungi pseudomonas of the present invention (Pseudomonas protegens) LK-313 derives from even
Making in Radix Pseudostellariae rhizosphere soil, be not originate from other habitats, rhizosphere colonization effect is preferable and safe and reliable, to Radix Pseudostellariae and after
Other crops of stubble (as crop is often planted in the Radix Pseudostellariae Genuine producing area such as Oryza sativa L., Semen sojae atricolor, Semen Maydis, vegetable) is all without the infection ability that causes a disease.
(3) the cultivation temperature of Antagonistic Fungi pseudomonas of the present invention (Pseudomonas protegens) LK-313
Degree ranges preferably from 4 DEG C～45 DEG C, and pH scope is 5～9, and the temperature and the pH scope that are suitable for growth are relatively wide, strong adaptability.
Accompanying drawing explanation
Fig. 1 is the main crop (A) and continuous cropping (B) Radix Pseudostellariae field growing situation comparison in difference.
Fig. 2 is the colonial morphology of the pathogen fusarium moniliforme that Radix Pseudostellariae plant diseases root is separated to. A: represent bacterium
Fall front；B: represent bacterium colony reverse side.
Fig. 3 is the opposite culture photograph of pseudomonas (Pseudomonas protegens LK-313) and fusarium moniliforme
Sheet. left planar is matched group (only inoculating fusarium moniliforme), and right planar center inoculation fusarium moniliforme, at peripheral 2.5cm
Inoculation Antagonistic Fungi.
Fig. 4 is the cellulose degradation merit rating of pseudomonas (Pseudomonas protegens LK-313).CK table
Show negative control, for pseudomonas fluorescens, not there is cellulose degradation ability；313 represent inoculation Antagonistic Fungi Pseudomonas
protegens LK-313。
Fig. 5 is pseudomonas (Pseudomonas protegens LK-313) optimum medium optimization.
Fig. 6 is that pseudomonas (Pseudomonas protegens LK-313) prevention and control fusarium moniliforme infects Radix Pseudostellariae group
Seedlings cultivating. Gm: represent and only inoculate fusarium moniliforme；313-Gm: represent inoculation Antagonistic Fungi LK-313 and fusarium moniliforme simultaneously.
Detailed description of the invention
Below in conjunction with specific embodiment, technical scheme is described in detail.Following example are used only for
The description and interpretation present invention, and do not constitute the restriction to technical solution of the present invention.
The screening of embodiment 1 Antagonistic Fungi pseudomonas (Pseudomonas protegens LK-313) and qualification thereof
1, the Antagonistic Fungi screening of Radix Pseudostellariae specialized form fusarium moniliforme
Antagonistic Fungi pseudomonas of the present invention (Pseudomonas protegens LK-313) is from continuous cropping Radix Pseudostellariae rhizosphere
In soil, screening obtains.
1.1 pseudomonas culture medium preparations
Compound method is such as pseudomonas selective medium (pseudomonas selective isolation agar, PSIA)
Under: weigh 20 g Soybean-casein digest agar culture mediums (soybean casein digest agar, SCD) (BD,
USA), add 495.5 mL distilled water heated and stirred, after fully dissolving, add 1 mL 0.1%(wt/vol) crystal violet storing solution,
121 DEG C of autoclave sterilization 15 min, when being cooled to about 50 DEG C, add 3.5 mL 5%(wt/vol in culture medium again) furan
Mutter nozzle pyridine storing solution, fully after mixing, rapid a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, i.e. prepare pseudomonas selective medium flat board after cooled and solidified.
Wherein, 0.1%(wt/vol) compound method of crystal violet storing solution is: weigh 0.1 g crystal violet (Sangon, on
Sea), it is dissolved in 100 mL distilled waters, room temperature preservation is standby；The compound method of Nitrofurantoin storing solution is: weigh 5 g furan appropriate
Because of (Sangon, Shanghai), join in 90 mL DMFs (Sangon, Shanghai) and fully dissolve, then be settled to
100 mL, room temperature keeps in Dark Place standby.
1.2 pseudomonass are cultivated
Weighing 10 g Radix Pseudostellariae rhizosphere soils, be dissolved in 90 mL sterilized water, fully vibration shakes up, and obtains 10-1Diluent.Take 10
mL 10-1In the sterilized water that dilution Soil Slurry cools down to another bottle 90 mL, fully vibration shakes up, and obtains 10-2Dilution
Liquid.Dilute successively 10-3Diluent, draws the soil dilution liquid of 60 μ L, and even spread is to pseudomonas selective medium
On, the lucifuge in 30 DEG C of constant incubators that is placed on cultivate 30 h.Select clear and legible in culture medium and the uniform single bacterium of growth
Falling, be forwarded on new pseudomonas selective medium, 30 DEG C are continued to cultivate about 30 h, are placed in of short duration guarantor in 4 DEG C of refrigerators
Deposit standby.
The Antagonistic Fungi screening of 1.3 Radix Pseudostellariae specialized form fusarium moniliformes
Potato dextrose medium (PDA) is formulated as follows: Rhizoma Solani tuber osi 200 g, after breaking into mashed potatoes with beater, adds suitable quantity of water
Boil rear timing 25 min, after it is cooled to non-scald on hand, crosses four layers of gauze.Glucose 30 g, fine jade is added after good for filtrate collection
Fat 15 g, heated and stirred mixes and adds water and is settled to subpackage after 1000 mL.Save backup after 115 DEG C of autoclaving 15 min.
During use, the culture medium that autoclave sterilization is crossed is reheated dissolving, treat that culture medium temperature drops to 55 DEG C of left sides
Plate quickly it is down flat time right (non-scald on hand).Cross bottom culture dish after culture medium fully solidifies frame, in the distance center of circle about 2.5
Inoculate the antibacterial of activated cultivation at cm, be placed in after lucifuge cultivates 48 h in 28 DEG C of constant incubators, at the centre bit of culture dish
Putting inoculation Radix Pseudostellariae specialized form fusarium moniliforme, be placed in lucifuge opposite culture a couple of days in 28 DEG C of constant incubators, Real Time Observation presses down
The size that has that it's too late of bacterium circle.After opposite culture 4 days, filter out a strain to Radix Pseudostellariae specialized form beading by observing inhibition zone size
Fusarium spp. has the antibacterial (as shown in Figure 3) of strong antagonistic effect, and this bacterial strain has been carried out preservation, and deposit number is CGMCC NO.
12121.The strong antagonistic strain screened is purified cultivation, and inclined-plane saves backup.
2, the Antagonistic Fungi of Radix Pseudostellariae specialized form fusarium moniliforme is identified
2.1 Antagonistic Fungis of the present invention (deposit number is: CGMCC NO. 12121) through microexamination it can be seen that this bacterium is
Corynebacterium, gram negative bacteria.For protuberance, moistening bacterium colony on LB solid medium.Meanwhile, the Physiology and biochemistry of this Antagonistic Fungi
Characteristic is: can decompose fructose, mannitol, ethanol, glucose, arginine, alanine and inositol, but do not reduce lactose, sucrose,
Xylose, glycerol and citric acid.Decomposable asymmetric choice net ammonium chloride, ammonium sulfate, potassium nitrate, ammonium nitrate and carbamide, but paddy ammonia can not be decomposed
Acid.Can not hydrolyze starch, can liquefy gelatin.The pH scope of normal growth is 5～9.Have stronger cellulose degradation ability (as
Shown in Fig. 4).
2.2 16s-23s rRNA gene interval PCR are identified
The strong antagonistic strain (deposit number is: CGMCC NO. 12121) filtered out is forwarded to 5 ml LB fluid mediums enter
After row amplification culture, (28 DEG C, 180 rpm) extract DNA.PCR expands 16s-23s rRNA gene interval, reflects for bacteria molecule
Fixed.Bacterial genomes DNA extraction method is as follows: bacterial genomes DNA extraction method is as follows: takes 1 mL and shakes pseudomonas overnight
Bacterium solution (28 DEG C, 180 rpm), 10000 rpm are centrifuged 5 min, remove supernatant, add 950 μ L TE buffer in precipitation thalline
Suspend precipitation, and adds 50 μ L 10% SDS solution and 5 μ L E.C. 3.4.21.64 (20mg/mL), mixing, and 37 DEG C of water-bath 1 h add
150 μ L 5 mol/mL NaCl solution and 150 μ L CTAB/NaCl solution (10% CTAB, 4.1% NaCl), mixing, 65 DEG C
Water-bath 20 min, adds isopyknic phenol chloroform isoamyl alcohol (volume ratio 25 24 1) solution and is stripped, and 12000 rpm are centrifuged
10 min, extract supernatant once with isopyknic chloroform/isoamyl alcohol (24/1) again, supernatant equal-volume isopropanol room temperature
Precipitating 30 min, 12000 rpm and be centrifuged 10 min, abandon supernatant, DNA precipitation is carried out with 70% ethanol, with 50 after natural air drying
μ L sterilized water dissolves.
Use 16s-23s rRNA gene interval PCR amplification technique that the pseudomonas of separation screening is carried out Molecular Identification.
Identify that primer sequence interval for 16s-23s is 1407f(5'-TTGTACACACCGCCCGTC-3') and 456r(5'-
CCTTTCCCTC ACGGTACTG-3').PCR amplification system (50 μ L) is: 25 μ L Taq PCR Master Mix (2 ×)
(TransGen, Beijing), 1.0 μ L upstream and downstream primer (10 μMs), 1 μ L DNA profiling (20 ng), 22 μ L sterilized water.PCR
Amplification program is: 94 DEG C of denaturation 5 min, 94 DEG C of degeneration 1 min, 60.5 DEG C of annealing 1 min, 72 DEG C of extension 90 sec, 35
Individual circulation, continues 72 DEG C and extends 10 min.The genetic fragment of amplification detects with 1% agarose gel electrophoresis, and uses Universal
DNA Purification Kit glue reclaims test kit (TIANGEN, Beijing) purification and reclaims purpose band, and serves sea raw work survey
Prelude checks order.Sequencing sequence uses BLAST instrument and ncbi database (Nucleotide collection(nr/nt))
Comparing, sequence is as shown in SEQ ID NO.3.This Antagonistic Fungi of molecular biology identification is pseudomonas, named
Pseudomonas protegens LK-313, deposit number is: CGMCC NO. 12121.
The fungistatic effect detection of embodiment 2 Antagonistic Fungi
Use PDA culture medium, at the distance culture dish center of circle 2.5 cm, inoculate the pseudomonas LK-313 of activated cultivation, be placed in
After lucifuge cultivates 48 h in 28 DEG C of constant incubators, inoculate Radix Pseudostellariae specialized form fusarium moniliforme in the center of culture dish,
It is placed in lucifuge opposite culture a couple of days, the formation of Real Time Observation inhibition zone in 28 ° of C constant incubators.Cultivate discovery in 4 days,
Pseudomonas protegens LK-313 can efficient antagonism Radix Pseudostellariae fusarium moniliforme mycelial growth (as shown in Figure 3).
Embodiment 3 pseudomonas (Pseudomonas protegens LK-313) optimum medium optimization
Preparing following culture medium to be optimized the most suitable growth culture medium of pseudomonas LK-313, culture medium is as follows: 1) full LB
Solution；2) 1/4 LB solution；3) 1/4LB+1/20MS；4) 1/4LB+1/40MS；5) 1/4LB+1/80MS；6) 1/4LB+1/20MS
+ 0.1wt.% brown sugar；7) 1/4LB+1/40MS+0.1wt.% brown sugar；8) 1/4LB+1/80MS+0.1wt.% brown sugar.
Wherein, MS culture medium (all without iron salt) formula is: 1) a great number of elements: 1900 mg/L KNO3、1650 mg/L
NH4NO3、370 mg/L MgSO4•7H2O、170 mg/L KH2PO4、440 mg/L CaCl2•2H2O；2) trace element: 22.3
mg/L MnSO4•4H2O、8.6 mg/L ZnSO4•7H2O、6.2 mg/L H3BO3、0.83 mg/L KI、0.25 mg/L
Na2MoO4•7H2O、0.025 mg/L CuSO4•5H2O、0.025 mg/L CoCl、2.6 mg/L H2O；3) Organic substance: 2.0
Mg/L glycine, 0.5 mg/L pyridoxine hydrochloride, 0.1 mg/L Tyiamine Hd element, 0.5 mg/L nicotinic acid, 100 mg/L fleshes
Acid；LB culture medium prescription is: tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L.
Concrete operation step is as follows: each culture medium of more than 5mL is respectively connected to the Antagonistic Fungi bacterium that equivalent (10 μ L) has activated
Liquid, be placed in 30 DEG C, on 180 rpm shaking tables concussion cultivate 10 h, take out culture test tube afterwards, bacterium solution fully shake up after in 600 nm
Light absorption value (0D is measured rapidly under wavelength600).Result shows: the cultivation of " 1/4LB+1/20MS+0.1wt.% brown sugar " this formula
Base is best suitable for the growth (as shown in Figure 5) of this Antagonistic Fungi.
Embodiment 4 Antagonistic Fungi prevention and control fusarium moniliforme infringement Radix Pseudostellariae tissue cultured seedling effect assessment
Preparation Radix Pseudostellariae tissue cultured seedling MS culture medium (MS+0.5 mg/L 6-BA+0.3 mg/L NAA+30 g/L sucrose
+ 7 g/L agar+8 g/L potato starch, pH=6.5), each tissue culture bottle adds 35 mL culture medium, through High Temperature High Pressure
Sterilizing, after solidification to be cooled, burns a ditch with tweezers in media surface, inoculates Radix Pseudostellariae seedling 2 strain in ditch side,
It is placed in after 25 DEG C of constant temperature group training rooms are cultivated 40 days, adds in ditch with the most suitable growth culture medium (1/4LB+1/20MS+
0.1wt.% brown sugar) the Antagonistic Fungi bacterium solution 300 μ L of overnight incubation, matched group adds equivalent LB culture medium and replaces, another at ditch
Side inoculation Radix Pseudostellariae specialized form fusarium moniliforme, continues during test to add pseudomonas LK-313 bacterium solution in ditch, sees
Examine the situation of infecting.Result shows, is added with in the test group of Antagonistic Fungi between pathogen and tissue cultured seedling, the growth quilt of fusarium moniliforme
Substantially suppression, only grows in ditch distal extent, and adds in the matched group of equivalent LB culture medium, and fusarium moniliforme growth is fast
Speed, it is possible to cross ditch, infect Radix Pseudostellariae tissue cultured seedling plant, cause tissue cultured seedling stem rot rotten, yellow leaf is withered, finally dead
(as shown in Figure 6).
As fully visible, the strain Antagonistic Fungi (preserving number is: CGMCC NO. 12121) that the present invention filters out is sick to Radix Pseudostellariae
Former bacterium has the strongest antagonism, it is possible to the effectively prevention and control fusarium moniliforme pathogen infringement to Radix Pseudostellariae, has in this field
Have broad application prospects.
The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent with
Modify, all should belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>University Of Agriculture and Forestry In Fujian
The bacterial strain of<120>one strain antagonism Radix Pseudostellariae specialized form fusarium moniliformes and application thereof
<130> 3
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213>artificial sequence
<400> 1
ttgtacacac cgcccgtc 18
<210> 2
<211> 19
<212> DNA
<213>artificial sequence
<400> 2
cctttccctc acggtactg 19
<210> 3
<211> 1047
<212> DNA
<213>artificial sequence
<400> 3
ttagccttgg aggtggtccc cccatattca gacaaggttt ctcgtgcccc gtcctactcg 60
atttcatgac taagagattt tcgcgtacag ggctatcacc cactatggcc gcactttcca 120
gagcgttccg ctaatctcaa agccacttaa gggctagtcc ccgttcgctc gccactacta 180
agggaatctc ggttgatttc ttttcctcag ggtacttaga tgtttcagtt cccctggttc 240
gcctcttgca cctatgtatt cagtacaaga taaccatctt atgatggctg ggttccccca 300
ttcagagatc tccggatcaa agtctgtttg ccgactcccc gaagcttatc gcaggctacc 360
acgtctttca tcgcctctga ctgccaaggc atccaccgta tgcgcttctt cacttgacca 420
tataacccca agcaatctgg ttatactatg aagacgacat tcgccgaaaa tttgcattct 480
taattaaaag agaactcaca aattttacct tagcctgatc cgttaccagt gaaagtaacg 540
ttcagtctat ctttctatca catacccaaa tttttaaaga acgatctaat caaaagacta 600
gaaatcaaca ttcaatgcga atgctcattt ctaagctttc agaagcagtt tatggtggag 660
ccaagcggga tcgaaccgct gacctcctgc gtgcaaggca ggcgctctcc cagctgagct 720
atggccccat aacaaaattg gtgggtctgg gcagattcga actgccgacc tcacccttat 780
caggggtgcg ctctaaccaa ctgagctaca gacccaattt cgagcttgta actgttagct 840
tggagctatc agcttggagc ttaaagctgc ttctatcgtc ttcttcaatg aatcaagcaa 900
ttcgtgtggg agcttatgaa gcagctgatg tcgtcgatta aggaggtgat ccagccgcag 960
gttcccctac ggctaccttg ttacgacttc accccagtca tgaatcacac cgtggtaacc 1020
gtcctcccga aggttagact agctact 1047
Claims (4)
1. the Antagonistic Fungi of a strain antagonism Radix Pseudostellariae specialized form fusarium moniliforme, it is characterised in that: described Antagonistic Fungi is false unit cell
Bacterium (Pseudomonas protegens) LK-313, is preserved in Chinese microorganism strain preservation management on January 28th, 2016
Committee's common micro-organisms center, culture presevation numbered CGMCC NO. 12121.
2. the growth medium of the Antagonistic Fungi of antagonism Radix Pseudostellariae specialized form fusarium moniliforme as claimed in claim 1, its feature
It is: its growth medium is the 1/4LB culture medium+1/20 MS culture medium+0.1wt.% brown sugar without iron salt.
The growth medium of the Antagonistic Fungi of preventing and treating Radix Pseudostellariae root rot the most according to claim 2, it is characterised in that: described
Without iron salt its formula of MS culture medium be: 1) a great number of elements: 1900 mg/L KNO3、1650 mg/L NH4NO3、370 mg/L
MgSO4•7H2O、170 mg/L KH2PO4、440 mg/L CaCl2•2H2O；2) trace element: 22.3 mg/L MnSO4•4H2O、
8.6 mg/L ZnSO4•7H2O、6.2 mg/L H3BO3、0.83mg/L KI、0.25 mg/L Na2MoO4•7H2O、0.025 mg/
L CuSO4•5H2O、0.025 mg/L CoCl、2.6 mg/L H2O；3) Organic substance: 2.0 mg/L glycine, 0.5 mg/L salt
Acid pyridoxol, 0.1 mg/L Tyiamine Hd element, 0.5 mg/L nicotinic acid, 100 mg/L creatines；Described LB culture medium is: pancreas egg
White peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L.
4. the Antagonistic Fungi of an antagonism Radix Pseudostellariae specialized form fusarium moniliforme as claimed in claim 1 is preventing and treating medicinal plants too
Application on son ginseng cultivating disease and soil-borne disease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610181836.5A CN105779342B (en) | 2016-03-28 | 2016-03-28 | The bacterial strain of one plant of antagonism radix pseudostellariae specialized form fusarium moniliforme and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610181836.5A CN105779342B (en) | 2016-03-28 | 2016-03-28 | The bacterial strain of one plant of antagonism radix pseudostellariae specialized form fusarium moniliforme and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105779342A true CN105779342A (en) | 2016-07-20 |
| CN105779342B CN105779342B (en) | 2019-04-02 |
Family
ID=56391956
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610181836.5A Active CN105779342B (en) | 2016-03-28 | 2016-03-28 | The bacterial strain of one plant of antagonism radix pseudostellariae specialized form fusarium moniliforme and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105779342B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113151044A (en) * | 2021-02-24 | 2021-07-23 | 贵州中医药大学 | Pseudomonas for preventing and treating diseases of radix pseudostellariae and separation, screening and identification method and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102199558A (en) * | 2010-08-06 | 2011-09-28 | 上海师范大学 | Culture method and applications of Pseudomonas aurantiaca |
| CN103509740A (en) * | 2013-08-13 | 2014-01-15 | 浙江大学 | Pseudomonas chlororaphis for preventing and treating crop fusarium disease and applications thereof |
| CN104877943A (en) * | 2015-06-11 | 2015-09-02 | 福建农林大学 | Antagonistic bacterium for controlling radix rehmannia root rot and application of antagonistic bacterium |
-
2016
- 2016-03-28 CN CN201610181836.5A patent/CN105779342B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102199558A (en) * | 2010-08-06 | 2011-09-28 | 上海师范大学 | Culture method and applications of Pseudomonas aurantiaca |
| CN103509740A (en) * | 2013-08-13 | 2014-01-15 | 浙江大学 | Pseudomonas chlororaphis for preventing and treating crop fusarium disease and applications thereof |
| CN104877943A (en) * | 2015-06-11 | 2015-09-02 | 福建农林大学 | Antagonistic bacterium for controlling radix rehmannia root rot and application of antagonistic bacterium |
Non-Patent Citations (2)
| Title |
|---|
| ALBAN RAMETTE等: "Pseudomonas protegens sp. nov., widespread plant-protecting bacteria producing the biocontrol compounds 2,4-diacetylphloroglucinol and pyoluteorin", 《SYSTEMATIC AND APPLIED MICROBIOLOGY》 * |
| 刘春静等: "串珠镰刀菌拮抗菌的分离筛选及其抑菌作用", 《粮食与饲料工业》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113151044A (en) * | 2021-02-24 | 2021-07-23 | 贵州中医药大学 | Pseudomonas for preventing and treating diseases of radix pseudostellariae and separation, screening and identification method and application |
| CN113151044B (en) * | 2021-02-24 | 2023-04-07 | 贵州中医药大学 | Pseudomonas for preventing and treating diseases of radix pseudostellariae and separation, screening and identification method and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105779342B (en) | 2019-04-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106434493B (en) | One plant of biological and ecological methods to prevent plant disease, pests, and erosion streptomycete and its application | |
| CN104877943B (en) | One plant preventing and treating Radix rehmanniae root rot Antagonistic Fungi and its application | |
| CN105695364B (en) | One plant of new Burkholderia cepacia and application | |
| CN104017744B (en) | The preparation method of the Pseudomonas chlororaphis agent of disease-resistant growth-promoting and purposes | |
| CN105586303B (en) | One plant of South Korea pseudomonad and its application | |
| CN102533617B (en) | Bacillus subtilis strain and application thereof | |
| CN104560827B (en) | A kind of biocontrol actinomycetes bacterial strain for preventing and treating tobacco bacterial wilt and its application | |
| WO2011032330A1 (en) | Antagonistic bacteria for preventing and treating bacterial wilt disease of continuously planted tobacco and microorganism organic fertilizer thereof | |
| CN102787090B (en) | Alfalfa root growth-promoting rhizobacteria MJM-5 and application of alfalfa root growth-promoting rhizobacteria MJM-5 | |
| CN102108339B (en) | Bacillus megaterium with capability of inducing stress resistance of soybeans and application thereof | |
| CN110358710A (en) | One plant of bacillus laterosporus and preparing the application in disease-resistant saline-alkali tolerant functional microorganism preparation | |
| CN107136122A (en) | A kind of biocontrol agent for preventing and treating the late blight of potato | |
| CN110218685A (en) | Biocontrol actinomycetes and application | |
| CN105296364A (en) | Penicillium oxalicum, preparation method and application thereof | |
| CN107142229B (en) | It is a kind of prevent and treat capsicum epidemic disease biocontrol actinomycetes bacterial strain and its application | |
| CN106701623A (en) | Bacillus atrophaeus antagonistic to lycium chinensis root rot and application of bacillus atrophaeus | |
| CN109971656B (en) | Ginger endogenetic trichoderma viride and application thereof | |
| CN104450560A (en) | Nematode-killing sphingosine bacillus strain and application thereof | |
| CN102108338B (en) | Cold-resistance-inducing Pseudomonas aeruginosa strain and application thereof | |
| CN106520566A (en) | Antagonistic fungus for preventing and treating bitter gourd wilt and application thereof | |
| CN109402009B (en) | Method for screening nitrogen-fixing blue algae antagonistic to rhizoctonia solani and application thereof | |
| CN102250781A (en) | Streptomyces strain for inhibiting harm of Phytophthora parasitica var. nicotianae and application thereof | |
| CN109355217B (en) | Bacillus subtilis and microorganism formulation and its application | |
| CN105779341B (en) | The Antagonistic Fungi of one plant of radix pseudostellariae root rot and application | |
| CN105820969A (en) | Bacillus amyliquefaciens strain and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |