CN104962501B - A kind of preparation and application of the bacterial strain, antagonist of anti-vegetable and fruit gray mold - Google Patents
A kind of preparation and application of the bacterial strain, antagonist of anti-vegetable and fruit gray mold Download PDFInfo
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Abstract
The invention discloses a kind of preparation and application of the bacterial strain, antagonist of anti-vegetable and fruit gray mold, the microbial strains are streptomycete（Streptomyces.sp）Strains A LS 045, deposit number are CGMCC No：10986.Antagonist preparation includes bacterial strain screening, and greenhouse pot culture activity is verified, prepared by bacterial strain Biology identification, biologically active agents, the verification of field activity and etc..Strains A LS 045 of the present invention is the excellent material for preventing vegetable and fruit grey mold disease biological agent, and production spore is abundant, strain liquid condition of culture is simple, preparation formulation is stablized, and is easy to industrialization amplification, and it is the excellent potentiality of green bio pesticide to have exploitation.
Description
Technical field
The invention belongs to actinomycetes strain and its preparations of antagonism vegetable and fruit pathogen, and in particular to streptomycete
（Streptomyces. sp）Bacterial strain and its antagonism preparation.
Background technology
Gray mold is the very wide fungal disease of a host range, generally occurs and flows in water fruits and vegetables breed selection
Row, gray mold are caused by the fungi of Deuteromycotina Hyphomycetes hyphomycetales Moniliaceae Botrytis, that is, infectedBotrytis cinereaCaused by Fungi Imperfecti Botrytis cinerea.Germ sporophore is several grows thickly for this, brown, and it is in 1 ~ 2 branch to have diaphragm, top,
The dense stalklet in top, size are 1452.5~3168.2 microns × 8.5~11.5 microns.Conidium ellipse is single to circle
Cell, nearly colourless, 4.2~10.5 microns × 3.5~7.5 microns of size.
Ash arrhizus bacteria mainly gets over the summer with invalid mycelia, sclerotium and conidium, is propagated by means of air-flow, rainwater or dew,
It is easily carried and propagates by farming operations, mildew generation is early, sprawling is fast, harm is heavy, loss is big.Vegetables in greenhouse gardening and fruit
In, such as tomato, cucumber, strawberry, grape be all highly prone to grey mold pathogen infection.Wherein, in vegetable variety, tomato is with Chinese olive
It falls ill heavier, germ infects remaining column cap or petal, expands to fruit, and pericarp is greyish white, has the mould layer of grey, water corruption to fall off.And
The female flower lost is opened in gray mold of cucumber intrusion, and water stain shape scab and the mould layer of taupe, floral organ softening, atrophy and corruption are generated in flower pedicle
Rotten, young melon rots to fall off.In fruit variety, strawberry fruit is caught an illness from remaining petal or close to the position on ground, leads to fruit
It rots, surface generates the mould layer of dense grey, causes diseased fruit rate 30% or so, serious up to 60% or more.And grape grey mould is not
But infect grape inflorescence and young fruit, cause its dehydration, atrophy, it is withered fall off, and also easy heavy losses in storing.These vegetables
The Production of fruit underproduction and loss, less serious case 30~40%, 50% or more severe one.It is current main using chemical pesticide prevention and control gray mold
Method.But chemical pesticide seriously pollutes environment, and the pesticide remained on agricultural product jeopardizes human health and life.It is made as biology
The microbial activity metabolite of agent, with prevention harmful organism, low toxicity, efficient, selectivity is strong, the residence time is short, environment is dirty
Small advantage is contaminated, the production and life style with the mankind are well mutually melted, and the Fermentation Engineering being gradually improved provides biological agent
The basis of industrialized production.
Invention content
The present invention is directed to by screening, culture and experiment, providing a kind of control vegetables and fruit gray mold has well
The antagonism gray mold microorganism of effectStreptomycesSp ALS-457 strains and its activating agent preparation method.
Above-mentioned purpose that the invention is realized by the following technical scheme：
（One）The bacterial strain of anti-vegetable and fruit gray mold
The bacterial strain is streptomycete bacterial strain（Streptomyces. sp）ALS-045, deposit number are CGMCC No：
10986。
（Two）Prepare bacterial strainStreptomyces. spThe method of ALS-045 antagonists
Include the following steps：
（1）Prepare spore suspension
It is inoculated with being inoculated on sterilized ISP2 agar slants from the production spore actinomyces of ALS-045 bacterial strains, 28 DEG C
Deionized water is added into inclined-plane after mycelium forms a large amount of spores by 4~7 d of culture, and lawn system is gently scraped with sterilizing bamboo stick
Standby spore suspension；
Above-mentioned slant medium ISP2 culture mediums are：4.0 g of yeast extract, 10.0 g of malt extract, 4.0 g of glucose,
20.0 g of agar, 1 L of tap water；
（2）50 ALS-045 bacterial strains are prepared into spore suspension, being inoculated in 100 L, sterilized liquid ISP2 is trained equipped with 60 L
In the fermentation tank for supporting base, 28 DEG C, 120 L/min ventilatory capacities, 150 rpm cultivate 3 d, wait for having a large amount of mycelium pellets to grow in culture solution
Stop culture；
（3）With step（2）Culture solution is seed liquor, and equipped with 350 L, sterilized liquid improves Gause I to culture transferring to 500 L
In the fermentation tank of culture medium, 28 DEG C, 120 L/min ventilatory capacities, 150 rpm cultivate 5 d, wait for there are a large amount of mycelium pellets in culture solution
Growth stops ventilation；
Aforesaid liquid improvement Gause I culture medium preparation method be：10.0 g of cornstarch, 10.0 g of sucrose, yeast
Medicinal extract 6.0 g, NaCl 0.5g, NaNO32.0 g, K2HPO4·3H2O 0.5 g, MgSO4· 7H2O 0.5 g, FeSO4·
7H20.02 g of O, tap water 1 L, pH 7.4~7.6；
（4）In 5% ratio to step（3）It is big that sterilized Amberlite XAD-16 are added in the cultivating system of fermentation tank
Macroporous adsorbent resin, 28 DEG C, 200 rpm adsorption treatments, 24 h, filtering fermentating liquid abandons filtrate, and mycelium is with resin mutually with 90% industry
Ethyl alcohol extracts 3~5 times, and extracting solution is concentrated under reduced pressure up to biological agent concentrate；
（5）Circulation step（2）~（4）, twice, it is brown yellow oil concentrate to amount to acquisition appearance for 500 L amplifications fermentation
9274 g, i.e. streptomycete bacterial strainStreptomyces. sp ALS-045。
（Three）Application of the antagonist in antagonism vegetable and fruit gray mold
Using including：Take streptomycete bacterial strain Streptomyces. sp ALS-045 dense by the 1.5% of dicyandiamide solution volume ratio
Dicyandiamide solution is added in contracting object, and the volume ratio of the dicyandiamide solution is：Ethyl alcohol：Tween80：Water=5：1：94, the ALS-045 concentrations
Object disperses evenly and stably under the solvent system.
The present invention has substantial advance and technique effect：
The present invention is inhibited with mycelium and the screening model of greenhouse pot culture different levels, and acquisition can wherein inhibit botrytis cinerea
Strongest microbial strains ALS-045 is grown, by molecular biology identification, ALS-045 is accredited asStreptomyces.
Sp bacterial strains.The bacterial strain is on June 17th, 2015 in " China Committee for Culture Collection of Microorganisms's common micro-organisms center "
Effective preservation is carried out（Address is：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode：100101）.Preserving number is：
CGMCC No.10986。
The present invention further withStreptomycesSp ALS-045 are starting strain, through cultivating,
Culture is extracted, strain liquid condition of culture directly utilizes Fermentation Engineering equipment, and production spore is abundant, and preparation formulation is steady
It is fixed.Formulated carried out field plot experiments have shown that：There is good result to prevention vegetable and fruit gray mold.
Strains A LS-045 of the present invention is the excellent material for preventing vegetable and fruit grey mold disease biological agent, is easy to industrialize
Amplification, it is the excellent potentiality of green bio pesticide to have exploitation.
Description of the drawings
Fig. 1 is strains A LS-04516S rRNA gene order Phylogenetic dendrograms.
Fig. 2 is strains A LS-045 scanning electron microscope aspect graphs.
Below in conjunction with specific implementation mode, the present invention will be further described.
Specific implementation mode
One, bacterial strain and screening
1, fermentation crude extract is prepared
87 plants of production spore actinomyces for being isolated from Ailaoshan Mountain National Nature Reserve, Yunnan, China soil are inoculated in ISP2 agar
On inclined-plane, which is 15 × 150 mm, 5 mL of slant medium volume.Every plant of bacterium is inoculated with 2 inclined-planes, wherein 1
Branch is spare inclined-plane, and in addition to spare inclined-plane, 5 are added into inclined-plane after mycelium forms a large amount of spores by 28 DEG C of 4~7d of culture
ML aseptic deionized waters, gently scraping lawn with sterilizing bamboo stick, to prepare spore suspension spare.
The preparation method of above-mentioned slant medium is：ISP2 culture mediums（g/L）：Yeast soaks 4.0 g, malt extract 10.0
G, 4.0 g of glucose, 20.0 g of agar, 1 L of tap water；PH is natural；121 DEG C of 20 min of sterilizing after preparing.
The spore suspension of every plant of bacterium is inoculated into the 250 mL triangles that Gause I culture medium is improved equipped with 50 mL liquid respectively
In bottle, every plant of bacterium is inoculated with 3 triangular flasks, while it is negative control that setting, which does not connect 3 triangular flasks of bacterium,.28 DEG C, 180 rpm conditions
Lower culture 7d；A large amount of mycelium pellets growth in test tube fluid nutrient medium is waited for, when culture solution color has significant change, in 2% ratio to each
The Amberlite XAD-16 macroporous absorbent resins that sterilized particle size range is 0.69 mm are added in culture bottle（Match good fortune in Beijing
Lai Bo Science and Technology Ltd.s）, 28 DEG C, 180 rpm stopping cultures in situ after adsorbing 24 h.
Aforesaid liquid improvement Gause I culture medium preparation method be：10.0 g of cornstarch, 10.0 g of sucrose, yeast
Medicinal extract 6.0 g, NaCl 0.5g, NaNO32.0 g, K2HPO4·3H2O 0.5 g, MgSO4· 7H2O 0.5 g, FeSO4·
7H20.02 g of O, tap water 1 L, pH 7.4-7.6.
It is unified to collect each shake flask culture, it is filtered with filter cloth, bacteria residue and resin are extracted using ethyl alcohol；Filtrate uses second
Acetoacetic ester extracts.Ethyl alcohol and ethyl acetate extract are concentrated under reduced pressure respectively to get fermentation crude extract.Crude extract of fermenting is 45
DEG C vacuum drying 24 h after, respectively accurately weigh 10 mg with 50 mL, 95% ethyl alcohol dissolve up to screen sample.Wherein, bacterium is derived from
Slag and resin are labeled as M samples using ethyl alcohol extraction sample；It is labeled as Y samples from culture filtered fluid ethyl acetate extraction sample.Later,
Into screening.
2, it screens
It is inoculated into the pathogenic bacteria obtained are detached from vegetables and fruit grey mold diseased plant on PDA agar slants, inclined-plane examination
Pipe specification is 15 × 150 mm, 5 mL of slant medium volume, is unified in 28 DEG C, cultivates 6~8 d under 400 nm illumination, waits for bacterium
After silk induction production spore, 5 mL sterile waters are added into inclined-plane, uses sterilizing bamboo stick gently to scrape lawn and prepares spore suspension as sieve
Sampling is spare.
Spore suspension is uniformly mixed with 200 mL PDA agar mediums of sterilizing postcooling to 45 DEG C, it is flat by 20 mL/
The amount of ware is poured into the sterilizing flat board that internal diameter is 9 cm, and the plate that carries disease germs is prepared into after natural cooling.Mycelia growth inhibition assay uses
Conventional cylinder plate method places sterilizing Oxford cup on each plate that carries disease germs, 200 microlitres of above-mentioned screenings is added with liquid-transfering gun in cup
Sample.Using 95% ethyl alcohol as negative control, often processing is repeated 3 times, 28 DEG C, cultivate 72 h under 400 nm illumination, is surveyed with crossing method
Determine antibacterial circle diameter.
The preparation method of the above PDA culture medium is（g/L）：200.0 g of potato, 20.0 g of sucrose, 20.0 g of agar, from
Water 1 L, pH are natural.Peeling potatoes are cut into block and boil 30 min, then use filtered through gauze, then sugaring, are supplied after dissolving
Water is to 1 L, 121 DEG C of 30 min that sterilize.
Experimental data shows：In all screening samples, sample is extracted from ALS-045 bacterial strains bacteria residue and resin ethyl alcohol（M
Sample）Show excellent antagonism botrytis cinerea growth ability.Antibacterial circle diameter respectively reaches 35 mm, 32 mm and 29 mm, and average 32
mm；And extract sample from ALS-045 strain culturing filtered fluid ethyl acetate（Y samples）Then without bacteriostatic activity.
Experimental data shows：Amberlite XAD-16 macroporous absorbent resins are suitable for handling the culture of ALS-045 bacterial strains
The subsequent processing of liquid.
Two, greenhouse pot culture confirmatory experiment
Test sample strains A LS-045 cultures are investigated under the conditions of greenhouse pot culture to main vegetables and fruit grey mold disease
Control effect.Comparison medicament is：50% iprodione wettable powder, by the production of Jilin power life agrochemical pesticide Co., Ltd.
After planting the consistent cucumber of 10 d or so growing ways, tomato, strawberry and grape cotyledon carry out protective effect and control for selection
Treatment effect measures.
Protective effect：By ALS-045 bacterial strains bacteria residue and resin ethyl alcohol test sample（M samples）It is uniformly applied to Huang with cotton swab
Melon, tomato, strawberry and grape cotyledon tow sides after 24 h, cut blade, petiole are wrapped in the cotton fully soaked through water
On.The gray mold germ fauna of 7 d or so will be cultivated, the bacteria cake of 5 mm of diameter is broken into card punch, bacterium downwards, is positioned over blade
It is each at positive nearly blade tip and nearly petiole to place 1 piece.Often processing sets 4 repetitions, often repeatedly 4 leaves, and sets clear water as blank control.
72 h of moisturizing culture in 20 DEG C of illumination box（Daily 12 h illumination, 12 h are dark）.
Therapeutic effect：The gray mold germ fauna of 7 d or so will be cultivated, the bacteria cake of 5 mm of diameter, bacterium face are broken into card punch
Downwards, each 1 piece of placement is positioned at the nearly blade tip of face of blade and nearly petiole.Often processing sets 4 repetitions, often repeatedly 4 leaves, and sets
Clear water is blank control.24 h of moisturizing culture in 20 DEG C of illumination box（Daily 12 h illumination, 12 h are dark）Afterwards, will
ALS-045 bacterial strains bacteria residue and resin ethyl alcohol test sample（M samples）Test plants cotyledon tow sides are uniformly smeared with cotton swab, wait for medicine
Blade is cut after liquid is dry, is wrapped on petiole with the cotton balls fully soaked through water, the moisturizing culture in 20 DEG C of illumination box.
Observe incidence：When blank control is fully fallen ill, each lesion diameter is measured using cross mensuration, by disease
Spot size carries out severity Scaling, is calculated by control effect formula.Grade scale is：0 grade：Disease-free spot；1 grade：Lesion diameter 0.1-
5.0 mm；3 grades：Lesion diameter 5.1-10.0 mm；5 grades：Lesion diameter 10.1-15.0 mm；7 grades：Lesion diameter 15.1-20.0
mm；9 grades：20.1 mm or more of lesion diameter.The control effect calculation formula is：
Disease index=×100
Control effect（%）=×100
The experimental results showed that：ALS-045 bacterial strains bacteria residue and resin ethyl alcohol test sample（M samples）To the temperature of test plant disease
Room potting control effect is good.Compared with being 87.68% with the therapeutic average preventive effect of comparison medicament, except the grape preventive effect that is averaged is
Outside 55.25%, M samples to cucumber, tomato, Botrytis cinerea disease therapeutic effect, average preventive effect is more than 70%.Wherein, for Huang
The therapeutic average preventive effect of melon is 72.23%, is 76.45% for the therapeutic average preventive effect of tomato, therapeutic average anti-for strawberry
Effect is 70.44%.Comparison medicament is suitable with the average preventive effect of M samples.
Except, which has good protective effect.Under protected mode model, for above-mentioned Four Plants, M samples are flat
Equal preventive effect reaches 80% or more.Wherein, it is 82.14% for the cucumber protectiveness preventive effect that is averaged, is averaged preventive effect for tomato protectiveness
It is 88.67%, is 84.90% for the strawberry protectiveness preventive effect that is averaged, is 80.43% for the grape protectiveness preventive effect that be averaged, and compares
Medicament protectiveness is averaged preventive effect as 84.24 %.
The above results show that：The good application prospect of ALS-045 bacterial strains.
Three, the molecular biology identification of purpose strains A LS-045
1, extracting genome DNA, amplification and sequencing
Purpose strains A LS-045 is seeded in sterilized 50 mL ISP2 fluid nutrient mediums, 180 rpm, under 28 °C
3 d are cultivated, the extraction, amplification and sequencing of genomic DNA are carried out.Wherein, the ingredient of ISP2 fluid nutrient mediums is（g/L）：Yeast
4.0 g of medicinal extract, 10.0 g of malt extract, 4.0 g of glucose, tap water 1 L, pH are naturally, prepare 20 min of rear 121 °C of sterilizings.
（1）Extract genomic DNA
Appropriate 3-5 mL cultures, 13000 rpm is taken to centrifuge 1 min, abandon culture medium.DNA is added and extracts lysate 300
Microlitre, be vortexed precipitation.65 DEG C of 30 min of water-bath.Isometric chloroform is added：Isoamyl alcohol（24:1）, vortex mixing, 13000 rpm
Centrifuge 5 min.Supernatant is taken, isometric isopropanol, mixing is added, 13000 rpm centrifuge 5 min.Supernatant is abandoned, 70% second is added
Alcohol mixing, 13000 rpm centrifuge 1 min.Ethyl alcohol is abandoned, centrifuge tube is inverted, dries.30 microlitres of ddH are added2O, dissolving DNA, -20
DEG C preserve.
（2）The amplification and sequencing of 16S rRNA genes
With following two primer amplification 16S rRNA genes：
27F：5′-AGA GTT TGA TCC TGG GCG CTC AG-3′；
1541R：5′-AAG GAG GTG ATC CAG CCG CAG-3′）.
Amplification system contains：
1 microlitre of DNA profiling（About 20~100 ng DNA）；
10 × PCR buffer solutions（100 mmol/L Tris-HCl, pH 8.3）；
500 mmol/L KCl；
15 mmol/L MgCl25 microlitres；
4 microlitres of dNTPs mixtures（Each dNTPs a concentration of 2.5 mmol/L, TaKaRa）；
1.25 U Taq enzymes（TaKaRa）；
Two primers（Final concentration of 0.4 μm of ol/L）Each 1 microlitre；
50 microlitres of aseptic deionized water.
PCR amplification instrument is GeneAmp PCR system（PE companies of the U.S.）, amplification program is：95°C 4 min；94°
C 1 min, 60 °C of 1 min, 72 °C of 1 min, 35 cycles；72°C 10 min.
PCR products are detected with 1% agarose gel electrophoresis, and the segment of 1500 bp sizes is then cut out with scalpel,
Use plastic recovery kit（WATSON Gel Extraction Mini Kit）It recycles and purifies target fragment.
（3）Sequence, terminator BigDye are measured with 377-96 sequencer of AB I PR ISM（Perkin-
Elmer）, sequencing primer is 27F and 1541R.
Sequencing reaction system：
About 1 microlitre of amplified production after purification（About 30 ng）,
2.5 microlitres of BigDye,
1 microlitre of primer（About 3.2 pmol/L）,
10 microlitres of aseptic deionized water.
Amplification program is：95 °C of 1 min, 56 °C of 1 min, 72 °C of 1 min, 35 cycles；72 °C, 5 min；4 °C of guarantors
It deposits.
By passing through PCR amplification and sequencing to ALS-045 bacterial strain 16S rRNA sequences, acquisition length is 886 effective 16S
RRNA gene base sequences.
2, the analysis of 16S rRNA genes base
The DNA sequence dna measured is through NCBI（National Center for Biotechnology Information）
The 16S rRNA gene orders that related species are obtained after BLAST engine search, are arranged with 1.8 softwares of ClustalX.It excludes
Base deletion site, with adjacent method phylogenetic tree construction.Distance matrix is calculated according to Kimura ' s two-parameter models,BootstrapIt examines and carries out 1000 sub-samplings.
As a result it shows：Positions of the purpose strains A LS-045 on phyletic evolution development tree is reliable（BootstrapValue>70）.
Line segment scale（0.005）Indicate the branch length of 0.5% sequence difference.On phyletic evolution development tree, strains A LS-045 and mark
Quasi- bacterial strainStreptomyces roseocinereus16S rRNA gene order maximum comparabilities reach 99. 94%（See Fig. 1）.
3. the strains A LS-045 scanning electron microscope of mesh is observed
ALS-045 bacterial strains are prepared into spore suspension（Method is the same as example 1）, take 200 microlitres of even spreads to having prepared ISP2
On agar plate（ISP2 medium components are shown in example 1）.Sterilized lid glass is entered with 30~45° angle oblique cutting on tablet later
Piece.Tablet cultivates 15 d in 28 DEG C of insulating boxs, wait for media surface it is a large amount of there is the mycelia of spore to spread to grow to lid glass
On piece can stop cultivating.Carefully the coverslip for being attached with mycelia growth is taken out with tweezers, 105 mm for being positioned over sterilizing are empty
It sets in culture dish, at room temperature after fully dry 5 d, carries out mycelium surface metal spraying, you can be scanned electron microscope sight
It examines.The model of electron microscope：Quanta 200（FEI Co., the U.S.）.
From scanning electron microscope in terms of result：Streptomyces microorganism characteristic feature is presented in strains A LS-045：Spore
The more a length of helical form of chain, spore are ellipsoid, and there is protrusion on surface（See Fig. 2）.It is observed under natural light, the bacterium early growth period bacterium
It falls surface and lark is gradually formed by whitewash red as growth time extends in ivory buff；Aerial hyphae is abundant, very thin；
Substrate mycelium is not broken, for purple yellow；The soluble purple uranidin of production, has the feature of typical streptomycete.But bacterium colony, the bacterium of the bacterium
Silk color and pigment generate situation and reference cultureStreptomyces roseocinereusIt has differences, it is still necessary to fixed kind.
In summary 16S rRNA genes and microorganism characteristic feature infer that strains A LS-045 belongs to streptomyces
（Streptomyces.sp）Bacterial strain.
Four, the preparation of strains A LS-045 biological agents
50 ALS-045 bacterial strains are prepared into spore suspension（Example 1）, it is inoculated in 100 L and 60 L sterilized liquids is housed
ISP2 culture mediums（ISP2 culture mediums preparation method is the same as example 3）Fermentation tank in（Fermentation tank model GUJS-100.Zhenjiang east
Bioengineering equipment and technology Co., Ltd）, 28 DEG C, 120 L/min ventilatory capacities, 150 rpm cultivate 3 d, wait in culture solution
There are a large amount of mycelium pellet growths to stop culture.Using the culture solution as seed liquor, its culture transferring to 500 L is equipped with 350 L sterilized solutions
The good Gause I culture medium of structural reform（Gause I culture medium preparation method is improved with example 1）Fermentation tank in（Fermentation tank model
For GUJS-500.Zhenjiang Oriental Bio-engineering Technology Co., Ltd）, 28 DEG C, 120 L/min ventilatory capacities, 150 rpm
5 d are cultivated, wait for there are a large amount of mycelium pellet growths to stop ventilation in culture solution.
It is added sterilized Amberlite XAD-16 macroporous absorbent resins into cultivating system in 5% ratio, 28 DEG C, 200
24 h of rpm adsorption treatments.Filtering fermentation liquor, filtrate abandon, and mycelium is mutually extracted 3-5 times with 90% industrial alcohol with resin, extraction
Liquid is concentrated under reduced pressure up to this biological agent concentrate.500 L amplifications fermentation carries out twice, amounting to and obtaining crude extract 9274 altogether
G, appearance are pale tan oil.
It is formulated for field plot bioorganism preparation by 1.5% proportional sampling, dicyandiamide solution volume ratio is：
Ethyl alcohol：Tween80：Water=5：1：94
Concentrate disperses evenly and stably under the solvent system.
Five, ALS-045 bacterial strains biological agent drug effect test of field zone
By medicine inspecting institute of the Ministry of Agriculture " pesticide field efficacy medicine test criterion " Selection experiment object, crop and kind, arable farming
And environmental condition.
Test medicine is：1.5% ALS-045 biological agents（It is developed by institute of microbiology of Yunnan University）.Comparison medicament
For：400 g/L pyrimethanils（Bayer Cropscience Co., Ltd produces）And 500 g/L iprodione suspending agents（Bayer crop section
Company produces）.
Application method is spray-on process.Spraying equipment is 16L-8AH type knapsack electric sprayers.
Corresponding spraying time is formulated according to each crop gray mold regularity of occurrence and development：At the beginning of disease, i.e., incidence does not surpass
Start first time dispenser when crossing 5% generation, it is hereafter primary at interval of 5~7 d, totally 3 times.Each reagent agent and clear water control are pressed
750 L/ hectares of dosage carries out.Crop is investigated by medicine inspecting institute of the Ministry of Agriculture " pesticide field efficacy medicine test criterion " drug effect computational methods
Control effect.
On January 7th, 2014 has carried out 1.5% ALS- on 2 6th, 2014, in Yunnan Province Xishan District, Kunming City United Town
045 biological agent prevents grey mould fruit rot of strawberry field control effectiveness test.Test result shows：1.5% ALS-045 biological agents（1800
G/ hectares of dosage groups）62.88% is reached to the control effect of grey mould fruit rot of strawberry；400 g/L pyrimethanils of comparison medicament（750
G/ hectares）With 500 g/L iprodione suspending agents（750 g/ hectares）Prevention μ μ μ effects to grey mould fruit rot of strawberry are respectively
81.79%、78.97%。
2 days to 2014 Septembers of August in 2014 1 day, have carried out 1.5% ALS- in Honghe state, Yunnan Province Jianshui County Qujiang River town
045 biological agent prevents grape grey mould field control effectiveness test.Test result shows：1.5% ALS-045 biological agents（2700
G/ hectares of dosage groups）69.82% is reached to the control effect of grape grey mould；400 g/L pyrimethanils of comparison medicament（750
G/ hectares）With 500 g/L iprodione suspending agents（750 g/ hectares）Control effect to grape grey mould is respectively 82.48%,
79.86%。
20 days to 2014 Septembers of August in 2014 18 days, have carried out 1.5% in Yunnan Province Kunming Jinning County Kunyang town
ALS-045 biological agents prevent gray mold of cucumber field control effectiveness test, and test result shows：1.5% ALS-045 biological agents
（1800 g/ hectares of dosage group）Control effect to gray mold of cucumber is respectively 71.17%；400 g/L pyrimethanils of comparison medicament are outstanding
Floating agent（750g/ hectares）With 500 g/L iprodione suspending agents（750 g/ hectares）Control effect to gray mold of cucumber is respectively
81.44%、80.15%。
Carry out 1.5% in Yunnan Province Kunming Jinning County Kunyang town on November 9,10 days to 2014 October in 2014
ALS-045 biological agents prevent graw mold of tomato field control effectiveness test, and test result shows：1.5% ALS-045 biological agents
（1800 g/ hectares of dosage group）63.69% is reached to the control effect of graw mold of tomato；400 g/L pyrimethanils of comparison medicament suspend
Agent（750 g/ hectares）With 500 g/L iprodione suspending agents（750 g/ hectares）Control effect to graw mold of tomato is respectively
76.08%、73.88%。
The above result shows that：Strains A LS-045 can be used as the excellent material of biological agent research and development, the biological agent prepared
Excellent results with prevention vegetable and fruit grey mold disease, can be developed further into as green bio pesticide.
Claims (3)
1. a kind of bacterial strain of anti-vegetable and fruit gray mold, it is characterized in that：The bacterial strain is streptomycete bacterial strain（Streptomyces.sp）ALS-045, deposit number are CGMCC No：10986.
2. the method for preparing antagonist using bacterial strain described in claim 1, includes the following steps：
（1）Prepare spore suspension
It is inoculated with being inoculated on sterilized ISP2 agar slants from the production spore actinomyces of ALS-045 bacterial strains, 28 DEG C of cultures 4
Deionized water is added into inclined-plane after mycelium forms a large amount of spores by~7 d, and gently scraping lawn with sterilizing bamboo stick prepares spore
Sub- suspension；
Above-mentioned slant medium ISP2 culture mediums are：4.0 g of yeast extract, 10.0 g of malt extract, 4.0 g of glucose, agar
20.0 g, 1 L of tap water；
（2）50 ALS-045 bacterial strains are prepared into spore suspension, 100 L is inoculated in and 60 L sterilized liquid ISP2 culture mediums is housed
Fermentation tank in, 28 DEG C, 120 L/min ventilatory capacities, 150 rpm cultivate 3 d, wait for having in culture solution a large amount of mycelium pellets growths to stop
Only cultivate；
（3）With step（2）Culture solution is seed liquor, and equipped with 350 L, sterilized liquid improves Gause I culture to culture transferring to 500 L
In the fermentation tank of base, 28 DEG C, 120 L/min ventilatory capacities, 150 rpm cultivate 5 d, wait for having a large amount of mycelium pellets to grow in culture solution
Stop ventilation；
Aforesaid liquid improvement Gause I culture medium preparation method be：10.0 g of cornstarch, 10.0 g of sucrose, yeast extract
6.0 g, NaCl 0.5g, NaNO32.0 g, K2HPO4·3H2O 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O
0.02 g, tap water 1 L, pH 7.4~7.6；
（4）In 5% ratio to step（3）Sterilized Amberlite XAD-16 macropores are added in the cultivating system of fermentation tank to inhale
Attached resin, 28 DEG C, 200 rpm adsorption treatments, 24 h, filtering fermentating liquid abandons filtrate, and mycelium is with resin mutually with 90% industrial alcohol
Extraction 3~5 times, extracting solution are concentrated under reduced pressure up to biological agent concentrate；
（5）Circulation step（2）~（4）, twice, it is brown yellow oil concentrate 9274 to amount to acquisition appearance for 500 L amplifications fermentation
G, the concentrate are containing streptomycete bacterial strain（Streptomyces. sp）The antagonist of ALS-045.
3. the application of antagonist prepared by method as claimed in claim 2 in antagonism vegetable and fruit botrytis cinerea.
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