CN104017744B - The preparation method of the Pseudomonas chlororaphis agent of disease-resistant growth-promoting and purposes - Google Patents
The preparation method of the Pseudomonas chlororaphis agent of disease-resistant growth-promoting and purposes Download PDFInfo
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Abstract
The invention discloses preparation method and the purposes of the Pseudomonas chlororaphis agent of a kind of disease-resistant growth-promoting；This bacterial strain is Pseudomonas chlororaphis (Psedunomonas chlororaphis) HT66CCTCC NO:M2013467.The main metabolites of this bacterial strain is azophenlyene 1 carboxylic acid and azophenlyene 1 Methanamide.By in this inoculation to microbiological culture media, obtain thalline and the metabolite of high concentration the most afterwards, can be prepared as microbial bacterial agent with material mixing such as Powdered Activated Carbon, Bentonite, bentonite, Kaolin, kieselguhr, zeolite and calcium carbonate, in finished product, number of viable is 1～8.5 × 109Cfu/mL.By flat board it is experimentally confirmed that described microbial inoculum has good prevention effect to rice sheath blight disease, cotton wilt, gaeumannomyces graminis, the pathogen of watermelon blight, the growth to Activities of Some Plants simultaneously also has good facilitation.
Description
Technical field
The invention belongs to biological pesticide and biological fertilizer field, relate to the microorganism formulation of disease prevention growth-promoting preparation method and
Application；It is specifically related to preparation method and the purposes of the Pseudomonas chlororaphis agent of a kind of disease-resistant growth-promoting.
Background technology
The result of study of FAO (Food and Agriculture Organization of the United Nation) shows, diseases and pests of agronomic crop natural loss rate more than 37%, serious shadow
Ring grain security and the safety of environmental ecology.According to investigations, the disease biological of China's common crops reaches 1600 kinds, causes near every year
The loss of 100000000000 RMB, seriously hinders the development of China's agricultural.At present, due to chemical pesticides various in agricultural production
A large amount of uses, number of types of pest and disease damage creates serious drug resistance to this kind of medicine, causes prevention effect continuous
Reduce.It is reported, the pest species of ten Nian Lai China outburst doubles above.Meanwhile, some pesticide are the most residual
Stay in soil, plant and water body, health is caused potential threat.When China's many agricultural byproducts export to foreign countries,
Usually meeting with external " green barrier " to close down because pesticide residues exceed standard, annual loss is up to 10,000,000,000 dollars, causes
The extensive concern of the whole society.
In the infectivity disease of crops, mainly there are fungal disease, bacterial disease, Disease and nematodiasis
Deng.Wherein, fungal disease is to be currently known the disease of most species in plant disease, accounts for the 80%-90% of disease species；And
Fungal disease can occur in each position of plant, and symptom type is the most most.
Rice sheath blight disease is also known as moire disease, and popular name spends sufficient stalk, rotten foot pestilence, features speckle.It is by Rhizoctonia solani Kuhn
(Rhizoctonia solani) infects and falls ill, and how to occur under high temperature, super-humid conditions.In Asia, America, Africa rice cultivation
Country generally occur.All there is distribution in Ge Dao district of China, but it is the most serious to cause harm in South Rice Region of China, causes setting percentage and mass of 1000 kernel to show
Writing and reduce, even plant example volt is withered, is one of Major Diseases in current Rice Production.Due to occurring area frequency wide, popular
Rate is high, and its caused loss is often beyond rice blast.
Watermelon blight is also known as dead arm, stem wilt, and its cause of disease is Fusarium spp. (Fusarium oxysporum
F.sp.niveum), belong to the fungus of Fungi Imperfecti, belong to Moniliales, Tuberculariaceae in soil the time-to-live up to 10
As long as Nian.Citrullus vulgaris all can be fallen ill from Seedling Stage to Adult plant, but the peak time of morbidity is the setting phase of blooming.In recent years, China
Watermelon blight occurs serious, causes a large amount of underproduction of Citrullus vulgaris, the general sick field underproduction 30%～40%, the severe disease Tian Zeke underproduction 80%
Below even have no harvest, produce to Citrullus vulgaris and cause serious economic loss.
Pythium ultimum (Pythium ultimum Trow) is initially isolated from the Herba Oenanthes Javanicae seedling of Britain's corruption, reports at present
Road is widely distributed, and it is not only perched in soil, the most extensively infects Semen sojae atricolor, Kidney bean, Semen Pisi sativi, Rhizoma Dioscoreae esculentae, Song Miao, coffee, Fructus Mali pumilae, Citrus chachiensis Hort.
Fructus Citri tangerinae, Fructus Persicae, Cotton Gossypii, Flos Chrysanthemi, Dahlia Pinnata Cav., Fructus Cucurbitae moschatae, Citrullus vulgaris, Caulis Sacchari sinensis, Herba Medicaginis, Fructus Lycopersici esculenties etc. more than 150 plant economic plants, cause Seedling withered, sudden
The multiple diseases such as, root-rot, foot are rotten, wither.
Plant rhizosphere growth-promoting antibacterial (plant growth-promoting rhizobacteria, PGPR) is a group field planting
In the closely-related rhizosphere bacteria of plant rhizosphere and plant roots.A large amount of greenhouses and field experiment result show, many PGPR bacterial strains
Show under greenhouse, field condition and significantly promote plant growing, prevent and treat soil-borne disease, improve plants against abiotic stress
The effects such as resistance, promotion geobiont reparation.PGPR is one of effective way of radical cure continuous cropping obstacles (continuous cropping is sick).
Pseudomonas is the gram negative bacteria that a class is distributed widely in nature, is also the common micro-of plant rhizosphere
Biotic population.Many pseudomonass can produce siderophore, antibiotic, the outer metabolite such as hydrolytic enzyme and hydrocyanic acid of born of the same parents, and protection is planted
Thing is from the infringement of pathogenic microorganism, inducing plant resistance, and it also can secrete the compound of indoles simultaneously, promotes that plant is raw
Long.As plant rhizosphere growth-promoting antibacterial, pseudomonas is widely studied by global scientist.The antibiotic that pseudomonas produces
Kind is a lot, including pyrrolnitrin (prorolnitrin, Prn), phenazine-1-carboxylic acid (phenazine-1-carboxylic
Acid, PCA), 2,4-diacetyl phloroglucinols (2,4-diacetylphloroglucinol, DAPG), pyoluteorin
(pyoluteorin, Plt), pyo (pyocyanin, PYO), fat polypeptide etc..Azophenlyene compounds can carry as electronics
Body, consumes the reducing substanceses such as NAD (P) H inside cell, produce simultaneously active oxygen (reactive oxygen species,
ROS), target cell is made to be poisoned to death.Therefore azophenlyene compounds has the antifungal activity of wide spectrum, and is not likely to produce drug resistance
Property.As can be seen here, greatly developing efficiently, while the chemical pesticide of low toxicity, screen and apply and can secrete azophenlyene compounds
Pseudomonas for the preventing and treating of controlling fungal diseases of crop be one economical, be conducive to having of sustainable development of agricultural production
Effect approach.
Summary of the invention
It is an object of the invention to overcome the chemical pesticide preventing and treating plant fungal disease in above-mentioned prior art to produce
Journey is seriously polluted, and in soil, water body and crops, residual quantity is high, and the problem such as the most serious drug resistance, it is provided that a kind of
The preparation method of the Pseudomonas chlororaphis agent of disease-resistant growth-promoting and purposes.The invention provides one and can prevent and treat various plants disease simultaneously
Pathogenic fungi also promotes the microbial strains Pseudomonas chlororaphis HT66 (Psedunomonas of plant growing
Chlororaphis), and utilize this bacterial strain to prepare microbial bacterial agent, and it is withered to utilize this bacterial strain to prevent and treat rice sheath blight disease, Citrullus vulgaris
Disease, the Radix Betae chrysanthemum speckle droop etc. of withering plant disease.
For achieving the above object, the most isolated and purified pseudomonas HT66 producing green pigment to a strain of inventor, to it
16S rDNA carries out PCR clone and checks order, and analyzes its evolutionary relationship, and result shows that this bacterium with the similarity of Pseudomonas chlororaphis is
99%.Through whole-cell fatty acid analysis and with Sherlock data base's comparison of MIDI company, this bacterium and environmental sample medium green
Similarity SI of pin pseudomonas is 0.343.
When described Pseudomonas chlororaphis HT66 grows on solid agar medium, bacterium colony is circular, neat in edge and rule
Then, surface wettability, smooth；In its 1～2 day grown, bacterium colony presents khaki, and at the 3rd～4 day, bacterium colony surface there will be relatively
The most bottle-green material.
Described Pseudomonas chlororaphis HT66 is after liquid culture 2 days, and bacterium solution ratio is denser, and presents the obvious colour of loess
Color；After standing overnight, the bottom of fermentation liquid there will be substantial amounts of green precipitate.Separated purification and qualification, this be precipitated as azophenlyene-
1-carboxylic acid and the mixture of azophenlyene-1-amide.
It is an object of the invention to be achieved through the following technical solutions:
First aspect, the present invention relates to a kind of Pseudomonas chlororaphis HT66 (Psedunomonas chlororaphis
HT66)CCTCC NO:M2013467。
Preferably, the partial sequence of the 16S rDNA of described Pseudomonas chlororaphis is as shown in SEQ ID NO.1.
It is further preferred that the partial sequence of the described 16S rDNA of Pseudomonas chlororaphis is the universal primer with antibacterial
27F and 1429R carries out obtaining through order-checking after PCR amplification, wherein the sequence of 27F and 1429R respectively as SEQ ID NO.2 with
Shown in SEQ ID NO.3.
Preferably, the main metabolites of described Pseudomonas chlororaphis is phenazine-1-carboxylic acid (phenazine-1-
Carboxylic acid, No. CAS is 2538-68-3, is called for short PCA) and azophenlyene-1-Methanamide (Phenazine-1-
Carboxamide, No. CAS is 550-89-0, is called for short PCN).
Preferably, described Pseudomonas chlororaphis can suppress the Rhizoctonia solani Kuhn in plant pathogenic fungi by flat board
(Rhizoctonia solani), Fusarium oxysporum (Fusarium oxysporum f.sp.niveum), Pythium ultimum
(Pythium ultimum Trow) and Folium Stevlae Rebaudianae septoria musiva (Septoria steviae Ishiba Yokoyama et
Tani)。
Second aspect, the present invention relates to the Pseudomonas chlororaphis agent of a kind of disease-resistant growth-promoting, with microbiological culture media as raw material,
Pseudomonas chlororaphis HT66 (Psedunomonas chlororaphis HT66) CCTCC NO:M2013467 trains through cell expansion
It is prepared after Yanging.
Preferably, the carrier of described microbial inoculum is Powdered Activated Carbon, Bentonite, bentonite, Kaolin, kieselguhr, zeolite, carbonic acid
One or more in calcium.
The third aspect, the present invention relates to the Pseudomonas chlororaphis agent of a kind of above-mentioned disease-resistant growth-promoting in preparing biological pesticide
Purposes.
Preferably, described biological pesticide is for preventing and treating rice sheath blight disease, watermelon blight, Stevia Rebaudiana Bertoni Leaf Spot Disease or Semen sojae atricolor, dish
Bean, Semen Pisi sativi, Rhizoma Dioscoreae esculentae, Song Miao, coffee, Fructus Mali pumilae, Citrus, Fructus Persicae, Cotton Gossypii, Flos Chrysanthemi, Dahlia Pinnata Cav., Fructus Cucurbitae moschatae, Citrullus vulgaris, Caulis Sacchari sinensis, Herba Medicaginis, Fructus Lycopersici esculenti
The biological pesticide of the root rot of crop.
Fourth aspect, microbial manure is being prepared in the Pseudomonas chlororaphis agent that the present invention relates to a kind of above-mentioned disease-resistant growth-promoting
In purposes.
Compared with prior art, there is advantages that
1, the pseudomonas for Biological control has pseudomonas fluorescens, Pseudomonas aeruginosa, pseudomonas putida, Amur
The kind such as pseudomonas and Pseudomonas chlororaphis；In addition to Pseudomonas aeruginosa and Pseudomonas chlororaphis, remaining pseudomonas
All can not produce azophenlyene compounds, therefore its biocontrol mechanism is the most different；Further, compared with Pseudomonas aeruginosa, green pin is false
Zymomonas mobilis does not has pathogenic report so far, and therefore its environmental friendliness, safe and reliable.
2, the Pseudomonas chlororaphis used in the present invention, during its wild strain liquid culture, azophenlyene-1-concentration of forma is up to
420mg/L, for the wild strain that azophenlyene-1-Methanamide yield is the highest in the world at present, therefore its biological and ecological methods to prevent plant disease, pests, and erosion is powerful.
3, the development cost of microbial inoculum of the present invention is low: the nutritional need of Pseudomonas chlororaphis is the highest, and stability is preferable, processes bacterium
Carrier and auxiliary agent that agent is selected are cheap, and production technology is simple.
4, the microbial inoculum of the present invention can effectively prevent and treat rice sheath blight disease, gaeumannomyces graminis, watermelon blight, cotton wilt
Deng fungal disease.
Accompanying drawing explanation
By the detailed description non-limiting example made with reference to the following drawings of reading, the further feature of the present invention,
Purpose and advantage will become more apparent upon:
Fig. 1 is the solid agar flat board cultural character schematic diagram of Pseudomonas chlororaphis HT66；
Fig. 2 is the phylogenetic analysis schematic diagram of Pseudomonas chlororaphis HT66；
The liquid chromatogram of Fig. 3 Pseudomonas chlororaphis HT66 metabolite；
Fig. 4 is the antibacterial experiment picture of Pseudomonas chlororaphis HT66, and wherein, the picture left above is rice banded sclerotial blight cause of disease bacterium, upper right
Figure is Stevia Rebaudiana Bertoni Leaf Spot Disease bacterium, and lower-left figure is Pythium ultimum, and bottom-right graph is withered germ of water-melon.
Detailed description of the invention
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.Following example will assist in this area
Technical staff be further appreciated by the present invention, but limit the present invention the most in any form.It should be pointed out that, general to this area
For logical technical staff, without departing from the inventive concept of the premise, it is also possible to make certain adjustments and improvements.These broadly fall into
Protection scope of the present invention.
The Pseudomonas chlororaphis HT66 (Psedunomonas chlororaphis HT66) of the present invention, this bacterial strain in
State's Type Tissue Collection (being called for short CCTCC) preservation, depositary institution address is: China. Wuhan. and Wuhan University, postcode is:
430072, preservation date is: on October 12nd, 2013, deposit number is: CCTCC NO:M2013467.
Embodiment 1
One, the acquisition of Pseudomonas chlororaphis HT66 and qualification
First in the rice terrace be allowed to dry water, choose a strain grow rice plant vigorous, without any lesions visible, remove
Oryza sativa L. bar stem portion and the topsoil of 5cm thickness, take Rhizosphere sampling with aseptic medicine spoon；Weigh 1 gram of soil sample, be placed in equipped with 50mL
In the 250mL triangular flask of KMB culture medium, and it is (eventually the denseest to add ampicillin (final concentration of 50 μ g/mL), chloromycetin wherein
Degree is 15 μ g/mL) and cycloheximide (final concentration of 100 μ g/mL) reduce the pollution of fungus in soil.Then triangular flask is put
28 DEG C of shaken cultivation 2h on the shaking table that rotating speed is 200rpm；Sampling, is coated on containing identical antibiotic after diluting 1000 times
On solid KMB flat board, the single bacterium colony obtained is carried out with asynchronous culture method the experiment of the former bacterium of the water resistant sheath and culm blight of rice, it is thus achieved that the highest
Antibacterial activity bacterial strain is Pseudomonas chlororaphis HT66, sees Fig. 1.
Fig. 1 is the solid agar flat board cultural character schematic diagram of Pseudomonas chlororaphis HT66.Bacterial strain HT66 trains at KMB solid
Support and after growing 12 hours on base, form milky bacterium colony, a diameter of about 1.5mm, circular, rat, smooth, relatively thickness,
Easily provoke, neat in edge；Along with the prolongation of incubation time, bacterium colony gradually becomes yellow；Examining under a microscope, antibacterial presents bar
Shape, 0.3 μm-0.7 μ m 1.0 μm-1.2 μm, Gram-negative, without spore.After 24 hours, bacterium colony surface engenders green
Color crystal, is phenazine-1-carboxylic acid and the mixture of azophenlyene-1-Methanamide.
Bacterial strain HT66 is cultivated in KMB culture medium to logarithmic growth latter stage, with bacterial genomes DNA rapid extraction reagent
Box (Shanghai Jierui Biology Engineering Co., Ltd's product) extracts its genomic DNA.With this genomic samples as template, use antibacterial
16s rDNA universal primer 27F (5 '-AGAGTTTGATCMTGGCTCAG-3 ') and 1429R (5 '-
TACGGHTACCTTGTTACGACTT-3 ') carry out PCR experiment, the 16s rDNA sequence obtained through order-checking after amplification, such as SEQ ID
Shown in NO.1.This sequence carries out in the nucleic acid database of NCBI Blast analysis, and it is micro-that result shows with HT66 very high homology
The biological overwhelming majority is Rhodopseudomonas, selects front 15 species, downloads its 16S rDNA sequence, use MEGA5 software building
Cladogram.Fig. 2 is the phylogenetic analysis schematic diagram of bacterial strain HT66.As shown in Figure 2, bacterial strain HT66 and Pseudomonas chlororaphis GP72 is same
The confidence level in source is 93, and therefore this bacterium of preliminary proof is a strain Pseudomonas chlororaphis.
Two, the whole-cell fatty acid analysis of Pseudomonas chlororaphis HT66
Use four zoning collimation methods, in the HT66 inoculation the most fully activated to KMB flat board, after 24h, use inoculation
Ring collects the thalline about 40mg in the 3rd region.By saponification, methylating, n-hexane extraction and neutralizing treatment obtain the full cell of HT66
The extraction sample of fatty acid, then uses the microorganism automatic identifying system of MIDI company to analyze the composition of fatty acid.Then make
Use covariance matrix analysis, principal component analysis and pattern recognition analysis technology, by the analysis result of gas chromatogram and MIDI company
Clinical microorganism data base and environmental microorganism data base's phase comparison, result is as shown in table 1.
Table 1.Sherlock MIS microorganism automatic identifying system is reported
| Data base | Index similarity | Strain name |
| RTSBA6 | 0.343 | Pseudomonas-chlororaphis/aureofaciens/aura |
| RCLIN6 | 0.228 | Pseudomonas-putida-biotype A |
As shown in Table 1, bacterial strain HT66 with the index similarity of Pseudomonas chlororaphis in environmental microorganism data base is
0.343, the confidence criteria higher than 0.25, therefore further demonstrating this bacterium is a strain Pseudomonas chlororaphis.
Three, the preparation method of Pseudomonas chlororaphis HT66 microbial inoculum
The preparation method of Pseudomonas chlororaphis HT66 microbial inoculum comprises the steps:
1, the activation of Pseudomonas chlororaphis strain: prepare LB or the KMB culture medium of solid respectively；Take out freezing
Pseudomonas chlororaphis HT66, activates 48 hours in 26～32 DEG C above inoculation solid medium, and then transferred species is to new solid training
Support on base, continue to pass on 2～3 times；
2, prepared by the seed of Pseudomonas chlororaphis: preparation liquid LB or KMB culture medium；The thalline of picking one ring activation,
Be inoculated in 50mL fluid medium (being loaded on 250mL triangular flask), be placed on the constant-temperature table of 26～32 DEG C concussion cultivate 10～
20 hours, shaking speed was 150～5000 revs/min.Then, this seed liquor is transferred to further the liquid of 1000～5000mL
In body culture medium, cultivate under the same terms, to obtain that substantial amounts of growth is vigorous and the seed of stalwartness；Wherein, described LB training
Support consisting of of base: containing tryptone 10 grams in every liter of culture medium, yeast extract 5 grams, 10 grams of sodium chloride, pH7.5；Solid is trained
Foster base is the agar powder of interpolation 1.2% in corresponding fluid medium.
Wherein, described King ' s B culture medium (KMB): containing tryptone 20 grams in every liter of culture medium, K2HPO40.392
Gram, glycerol 15 milliliters, MgSO40.732 gram, pH7.2；Solid medium be in corresponding fluid medium add 1.2～
The agar powder of 1.5%.
3, the fermentation culture of Pseudomonas chlororaphis: the liquid LB of preparation large volume or KMB culture medium, loads in fermentation tank
In 121 DEG C of sterilizings 20 minutes；When fermentation jar temperature is cooled to 26～32 DEG C, the ratio with 1% accesses Pseudomonas chlororaphis
Seed liquor；Then at 26～32 DEG C, air agitation is cultivated, and fermentation tank rotating speed of agitator is 150～300 revs/min, ventilation ratio
For 1:1 (VVM), fermentation time is 30～40 hours.
The composition of fermentation medium there is no particular/special requirement, and wherein carbon source is starch, glucose, sucrose or glycerol, nitrogen source
Can be peptone, bean cake, meat extract, Semen Maydis pulp etc., additionally add the inorganic salts such as appropriate phosphate and magnesium sulfate, fermentation liquid
PH value is maintained at 7.0～8.0.
4, carrier is added: weigh appropriate Powdered Activated Carbon, Bentonite, bentonite, Kaolin, kieselguhr, zeolite or carbonic acid
The materials such as calcium, with 5～50% mass ratio add in the fermentation liquid of Pseudomonas chlororaphis, and fully stand-by after mixing.
Four, the Methanogenesis of Pseudomonas chlororaphis HT66
On flat board after fully activation HT66, it is inoculated in the 250mL conical flask containing 50mL KMB culture medium,
180rpm, after 28 DEG C are cultivated 16h, is transferred to the 2L conical flask containing 500mL KMB culture medium, 180rpm, cultivates 48h for 28 DEG C.
Timing sampling 1mL, with the salt acid for adjusting pH value of 6mol/L for 2.0, uses isopyknic ethyl acetate repeatedly to extract 3 fermentations
Liquid.Merge organic solvent, use Rotary Evaporators and vacuum drying oven to be fully evaporated sample, sample is redissolved in the first of 5mL
Alcohol (chromatographic grade).Using the reverse performance liquid chromatographic column of C18 (46 × 250mm, 5 μm, WondaSil, Japan) to analyze, flow phase
Consist of: 0-2min, 8% acetonitrile-92%25mM ammonium acetate；2-20min, acetonitrile concentration rises to 60% from 8%, ammonium acetate concentration
40% is dropped to from 92%；20-21 minute, 8% acetonitrile-92%25mM ammonium acetate, ultraviolet detection wavelength was 254nm, sets post
Temperature is 30 DEG C.The liquid chromatogram of Pseudomonas chlororaphis HT66 metabolite is shown in Fig. 3.
(phenazine-1-carboxylic acid, No. CAS is 2538-68-3, letter from the figure 3, it may be seen that phenazine-1-carboxylic acid
Claim PCA) and the reservation of azophenlyene-1-Methanamide (Phenazine-1-carboxamide, No. CAS is 550-89-0, is called for short PCN)
Time is respectively 9.532 minutes and 17.217 minutes.
Embodiment 2
The present embodiment is to prepare Pseudomonas chlororaphis agent on the basis of embodiment 1 and it is carried out biological characteristis；Tool
Body is as follows:
One, the preparation of microbial inoculum
The KMB solid of preparation solid and fluid medium, inoculate solid by the Pseudomonas chlororaphis HT66 of-80 DEG C of freezings
In body culture medium, activate 48 hours in 28 DEG C；Then transferred species is on new solid medium, continues to pass on 2 times；Then picking one
The thalline of ring activation, is inoculated in 50mL fluid medium (being loaded on 250mL triangular flask), is placed on the constant-temperature table of 28 DEG C concussion
Cultivating 16 hours, shaking speed is 180 revs/min.Then, this seed liquor is transferred to further the fluid medium of 1000mL
In, cultivate under the same terms；Finally prepare higher volume of liquid KMB culture medium, load in fermentation tank in 121 DEG C of sterilizings 20
Minute；When fermentation jar temperature is cooled to 28 DEG C, the ratio with 1% accesses the seed liquor of Pseudomonas chlororaphis；Then in 28 DEG C
Lower air agitation is cultivated, and fermentation tank rotating speed of agitator is 250 revs/min, and ventilation ratio is 1:1 (VVM), and fermentation time is 36 little
Time, and sampling and measuring bacterium solution OD value and azophenlyene-1-amide yield at regular intervals.Finally with gradient dilution method, measure final
Thalline quantity in unit volume fermentation liquid.Under this condition, the cell concentration of Pseudomonas chlororaphis HT66 is 7.9 × 109cfu/
mL.According to standard curve, azophenlyene-1-Methanamide yield reaches 424.87mg/L.
Two, the mensuration of heteroauxing
The assay method of plant growth-promoting factor indole-3-acetic acid (IAA) mainly according to Bric J.M. (Bric J.M.,
1991) method et al. detects after improving.Substantially step is as follows: fully activate HT66 bacterial strain in KMB culture medium, by bacterium
Single bacterium colony toothpick of strain HT66 is seeded in containing 5mM L-Trp, the LB culture medium center of 0.06%SDS and 1% glycerol
The heart.Use and cover on bacterium colony surface with Whatman1 filter paper, constant temperature culture 3 days in 28 DEG C of incubators.Drip at filter paper center
Salkowski ' s reagent (0.5mM FeCl3, 35%HClO4) process, HT66 bacterium colony position and near immediately
Redness circle occurs, illustrates that HT66 can produce plant growth-promoting factor indole-3-acetic acid.
Three, the antifungal experiment of bacterial strain
The Rhizoctonia solani Kuhn of diameter 8mm cultivated 3 days, ultimate corruption is accessed in potato dextrose medium flat board central authorities
Mould, Stevia Rebaudiana Bertoni Leaf Spot Disease bacterium and withered germ of water-melon pathogen mycelia block.Symmetrically placed a piece of diameter 8mm in each culture dish
Sterilizing filter paper, filter paper centre distance central authorities mycelia block 25mm.Take the HT66 microbial inoculum sample of above-mentioned preparation, dilute 100 times,
Filter paper accesses 10 μ L dilution of bacteria.The antibacterial experiment of every kind of pathogenic fungi carries out three parallel flats, and this is put down
Plate is placed in 28 DEG C of constant incubators cultivation 5d, measures inhibition zone radius.
Fig. 4 is the antibacterial experiment picture of Pseudomonas chlororaphis HT66.Wherein, the picture left above is rice banded sclerotial blight cause of disease bacterium, upper right
Figure is Stevia Rebaudiana Bertoni Leaf Spot Disease bacterium, and lower-left figure is Pythium ultimum, and bottom-right graph is withered germ of water-melon.From in Fig. 4, green pin vacation list
Rhizoctonia solani Kuhn, Pythium ultimum, Stevia Rebaudiana Bertoni Leaf Spot Disease bacterium and withered germ of water-melon pathogen are had significantly by born of the same parents bacterium HT66
Inhibitory action.
Above the specific embodiment of the present invention is described.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can make various deformation or amendment within the scope of the claims, this not shadow
Ring the flesh and blood of the present invention.
Claims (8)
1. a Pseudomonas chlororaphis, it is characterised in that described Pseudomonas chlororaphis is Pseudomonas chlororaphis (Psedunomonas
Chlororaphis) HT66 CCTCC NO:M 2013467；The partial sequence of the 16S rDNA of described Pseudomonas chlororaphis is such as
Shown in SEQ ID NO.1.
2. Pseudomonas chlororaphis as claimed in claim 1, it is characterised in that the portion of the described 16S rDNA of Pseudomonas chlororaphis
Sub-sequence is to carry out obtaining through order-checking after PCR amplification with universal primer 27F and 1429R of antibacterial, wherein 27F and 1429R
Sequence is respectively as shown in SEQ ID NO.2 and SEQ ID NO.3.
3. Pseudomonas chlororaphis as claimed in claim 1, it is characterised in that the main metabolites of described Pseudomonas chlororaphis
For phenazine-1-carboxylic acid and azophenlyene-1-Methanamide；No. CAS of described phenazine-1-carboxylic acid is 2538-68-3, described azophenlyene-1-first
No. CAS of amide is 550-89-0.
4. Pseudomonas chlororaphis as claimed in claim 1, it is characterised in that described Pseudomonas chlororaphis can flat board suppression plant
Rhizoctonia solani Kuhn (Rhizoctonia solani) in pathogenic fungi, Fusarium oxysporum (Fusarium oxysporum f.
Sp. niveum), Pythium ultimum (Pythium ultimum Trow) and Folium Stevlae Rebaudianae septoria musiva (Septoria steviae
Ishiba Yokoyama et Tani)。
5. the Pseudomonas chlororaphis agent of a disease-resistant growth-promoting, it is characterised in that with microbiological culture media as raw material, such as claim
Pseudomonas chlororaphis described in 1 is prepared after cell expansion is cultivated.
The Pseudomonas chlororaphis agent of disease-resistant growth-promoting the most as claimed in claim 5, it is characterised in that the carrier of described microbial inoculum is powder
One or more in end activated carbon, Bentonite, bentonite, Kaolin, kieselguhr, zeolite, calcium carbonate.
7. the Pseudomonas chlororaphis agent of a disease-resistant growth-promoting as claimed in claim 5 purposes in preparing biological pesticide, its
Being characterised by, described biological pesticide is for preventing and treating rice sheath blight disease, watermelon blight, Stevia Rebaudiana Bertoni Leaf Spot Disease or Semen sojae atricolor, Kidney bean, pea
Bean, Rhizoma Dioscoreae esculentae, Song Miao, coffee, Fructus Mali pumilae, Citrus, Fructus Persicae, Cotton Gossypii, Flos Chrysanthemi, Dahlia Pinnata Cav., Fructus Cucurbitae moschatae, Citrullus vulgaris, Caulis Sacchari sinensis, Herba Medicaginis, Fructus Lycopersici esculenti crop
The biological pesticide of root rot.
8. the Pseudomonas chlororaphis agent of a disease-resistant growth-promoting as claimed in claim 5 purposes in preparing microbial manure.
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