CN104962501A - Preparation and application of bacterial strain resistant to gray mould of vegetables and fruits and antagonist - Google Patents
Preparation and application of bacterial strain resistant to gray mould of vegetables and fruits and antagonist Download PDFInfo
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Abstract
The invention discloses preparation and application of a bacterial strain resistant to gray mould of vegetables and fruits and an antagonist, wherein a microbiological strain is streptomyces.sp ALS-045 with the preservation number of CGMCC No:10986. A preparation method of the antagonist comprises the following steps: screening a strain, verifying greenhouse potting activity, carrying out biological assay on the bacterial strain, preparing a biological active agent, and carrying out field activity verification. The bacterial strain ALS-045 is an excellent biological agent material for preventing and treating the gray mould of the vegetables and fruits, abundant spores are produced, bacterial strain liquid culture conditions are simple, a dosage form of a preparation is stable, industrialized enlargement is easy to implant, and the bacterial strain ALS-045 has great potential of being developed into green biopesticide.
Description
Technical field
The invention belongs to the preparation of actinomycetes strain and antagonism vegetable and fruit pathogenic bacteria thereof, be specifically related to streptomycete (
streptomyces. sp) bacterial strain and antagonism preparation thereof.
Background technology
Gray mold is the fungal disease that a host range is very wide, in fruit, vegetables breed selection generally occur and popular, gray mold causes by the fungi of Deuteromycotina hyphomycetes hyphomycetales Moniliaceae Staphlosporonites, namely infects
botrytis cinereacaused by imperfect fungi Botrytis cinerea.Germ sporophore is several grows thickly for these, and brown, has barrier film, and top is 1 ~ 2 branch, the close raw stalklet in top, and size is 1452.5 ~ 3168.2 microns × 8.5 ~ 11.5 microns.Conidium is oval to circular, unicellular, closely colourless, size 4.2 ~ 10.5 microns × 3.5 ~ 7.5 microns.
Ash arrhizus bacteria mainly gets over the summer with invalid mycelia, sclerotium and conidium, propagates by means of air-flow, rainwater or dew, is easily carried propagation by farming operation, and mildew occurs early, it is fast to spread, harm is heavy, loss is large.In the vegetables and fruit of greenhouse gardening, as tomato, cucumber, strawberry, grape etc. are all very easily subject to grey mold pathogen infection.Wherein, in vegetable variety, tomato is heavier with Chinese olive morbidity, and the column cap that infection process is residual or petal, expand to fruit, pericarp is greyish white, and have the mould layer of grey, water corruption comes off.And gray mold of cucumber invade open the female flower lost, produce water stain shape scab and the mould layer of beige at Hua Di, floral organ deliquescing, atrophy and rot, young melon rots to come off.In fruit variety, strawberry fruit is caught an illness from residual petal or near the position on ground, causes fruit rot, and surface produces the dense mould layer of grey, causes diseased fruit rate about 30%, seriously can reach more than 60%.And grape grey mould not only infects grape inflorescence and young fruit, cause its dehydration, atrophy, withered to come off, and also easy heavy losses in storage.These vegetable and fruit production underproduction and losses, the lighter 30 ~ 40%, severe one more than 50%.Using chemical pesticide prevention and control gray mold is current main method.But chemical pesticide serious environment pollution, the agricultural chemicals remained on agricultural-food jeopardizes human health and life.As the microorganism active meta-bolites of biotechnological formulation, there is control harmful organism, low toxicity, the advantage that efficient, selectivity is strong, the residence time is short, environmental pollution is little, well melt mutually with the production of the mankind and mode of life, and the fermentation engineering be gradually improved provides the basis of biotechnological formulation suitability for industrialized production.
Summary of the invention
The present invention is intended to by screening, cultivates and test, provides a kind of antagonism gray mold microorganism controlling to have good result to vegetables and fruit gray mold
streptomyces. sp ALS-457 bacterial classification and promoting agent preparation method thereof.
The present invention is achieved through the following technical solutions above-mentioned purpose:
(1) bacterial strain of anti-vegetable and fruit gray mold
Described bacterial strain is streptomycete bacterial strain
streptomyces.
spaLS-045, deposit number is CGMCCNo:10986.
(2) bacterial strain is prepared
streptomyces.
spthe method of ALS-045 antagonist
Comprise the following steps:
(1) spore suspension is prepared
The product spore actinomycetes deriving from ALS-045 bacterial strain are inoculated in and inoculate on the ISP2 agar slant of sterilizing, cultivate 4 ~ 7 d, formed after a large amount of spore until mycelium, in inclined-plane, add deionized water, gently scrape lawn with sterilizing bamboo let and prepare spore suspension for 28 DEG C;
Above-mentioned slant medium ISP2 substratum is: yeast extract 4.0 g, malt extract 10.0 g, glucose 4.0 g, agar 20.0 g, tap water 1 L;
(2) 50 ALS-045 bacterial strains are prepared spore suspension, be inoculated in 100 L and be equipped with in the fermentor tank of 60 L sterilising liq ISP2 substratum, 28 DEG C, 120 L/min air flows, 150 rpm cultivate 3 d, treat that namely have a large amount of mycelium pellet to grow in nutrient solution stops cultivating;
(3) with step (2) nutrient solution for seed liquor, culture transferring is equipped with in the fermentor tank of 350 L sterilising liq improvement Gause I substratum to 500 L, 28 DEG C, 120 L/min air flows, 150 rpm, cultivate 5 d, treat that namely have a large amount of mycelium pellet to grow in nutrient solution stops ventilation;
The compound method of aforesaid liquid improvement Gause I substratum is: W-Gum 10.0 g, sucrose 10.0 g, yeast extract 6.0 g, NaCl 0.5g, NaNO
32.0 g, K
2hPO
43H
2o 0.5 g, MgSO
47H
2o 0.5 g, FeSO
47H
2o 0.02 g, tap water 1 L, pH 7.4 ~ 7.6;
(4) in the culture system of step (3) fermentor tank, sterilized Amberlite XAD-16 macroporous adsorbent resin is added in 5% ratio, 28 DEG C, 200 rpm adsorption treatment 24 h, filtering fermentating liquid, abandon filtrate, mycelium and resin-phase 90% industrial alcohol extract 3 ~ 5 times, and namely extracting solution concentrating under reduced pressure obtains biotechnological formulation enriched material;
(5) circulation step (2) ~ (4), 500 L amplify fermentation twice, and amounting to acquisition outward appearance is brown yellow oil enriched material 9274 g, i.e. streptomycete bacterial strain
streptomyces. spaLS-045.
(3) application of antagonist in antagonism vegetable and fruit gray mold
Application comprises: get streptomycete bacterial strain Streptomyces. sp ALS-045 enriched material by 1.5% of solvent system volume ratio and add solvent system, the volume ratio of this solvent system is: ethanol: Tween80: water=5:1:94, and described ALS-045 enriched material is uniformly dispersed stable under this solvent systems.
The present invention has substantial advance and technique effect:
The present invention suppresses and the screening model of greenhouse pot culture different levels with mycelium, and obtain the microorganism strains ALS-045 that botrytis cinerea growth wherein can be suppressed the strongest, through molecular biology identification, ALS-045 is accredited as
streptomyces. sp bacterial strain.This bacterial strain on June 17th, 2015 " China Committee for Culture Collection of Microorganisms's common micro-organisms " center " has carried out effective preservation, and (address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode: 100101).Preserving number is: CGMCC No.10986.
The present invention further with
streptomyces. sp ALS-045 is starting strain, through cultivating,
Extract culture, strain liquid culture condition directly utilizes fermentation engineering equipment, and produce spore and enrich, preparation formulation is stablized.Prove through preparing the field plot experiment carried out: to control vegetable and fruit gray mold, there is good result.
Strains A LS-045 of the present invention is the excellent material of control vegetable and fruit grey mold disease biotechnological formulation, is easy to industrialization and amplifies, have the excellent potentiality being developed as green bio agricultural chemicals.
Accompanying drawing explanation
Fig. 1 is strains A LS-04516S rRNA gene order Phylogenetic dendrogram.
Fig. 2 is strains A LS-045 scanning electronic microscope aspect graph.
Below in conjunction with embodiment, the present invention will be further described.
Embodiment
One, bacterial strain and screening
1, preparation fermentation crude extract
Be inoculated on ISP2 agar slant by the 87 strains product spore actinomycetes be separated from Ailaoshan Mountain National Nature Reserve, Yunnan, China soil, this slant tube specification is 15 × 150 mm, slant medium volume 5 mL.Every strain bacterium inoculates 2 inclined-planes, and wherein 1 is inclined-plane for subsequent use, cultivates 4 ~ 7d, is formed after a large amount of spore, except inclined-plane for subsequent use, add 5 mL aseptic deionized waters in inclined-plane until mycelium for 28 DEG C, and gently scraping lawn with sterilizing bamboo let, to prepare spore suspension for subsequent use.
The compound method of above-mentioned slant medium is: ISP2 substratum (g/L): yeast soaks 4.0 g, malt extract 10.0 g, glucose 4.0 g, agar 20.0 g, tap water 1 L; PH nature; Prepare rear 121 DEG C of sterilizing 20 min.
The spore suspension of every strain bacterium is inoculated in the 250 mL triangular flasks that 50 mL liquid improvement Gause I substratum are housed respectively, and every strain bacterium inoculates 3 triangular flasks, and arrange 3 triangular flasks not connecing bacterium is negative control simultaneously.28 DEG C, cultivate 7d under 180 rpm conditions; Treat a large amount of mycelium pellet growth in test tube liquid nutrient medium, when nutrient solution color has a considerable change, in each culturing bottle, the Amberlite XAD-16 macroporous adsorbent resin (Beijing Sai Fulaibo Science and Technology Ltd.) that sterilized particle size range is 0.69 mm is added, 28 DEG C, 180 rpm original positions adsorb after 24 h and stop cultivating in 2% ratio.
The compound method of aforesaid liquid improvement Gause I substratum is: W-Gum 10.0 g, sucrose 10.0 g, yeast extract 6.0 g, NaCl 0.5g, NaNO
32.0 g, K
2hPO
43H
2o 0.5 g, MgSO
47H
2o 0.5 g, FeSO
47H
2o 0.02 g, tap water 1 L, pH 7.4-7.6.
The each shake flask culture of unified collection, filters with filter cloth, and bacterium slag and resin adopt extraction using alcohol; Filtrate adopts extraction into ethyl acetate.Ethanol and ethyl acetate extract concentrating under reduced pressure respectively, obtains fermentation crude extract.Fermentation crude extract, after 45 DEG C of vacuum-drying 24 h, accurately takes 10 mg, 50 mL 95% dissolve with ethanol separately and namely obtains screening sample.Wherein, deriving from bacterium slag and resin adopts extraction using alcohol sample to be labeled as M sample; Derive from cultivation filtered liquid ethyl acetate extraction sample and be labeled as Y sample.Afterwards, screening is entered.
2, screen
Be inoculated on PDA agar slant by being separated the pathogenic bacterium obtained from vegetables and fruit grey mold diseased plant, this slant tube specification is 15 × 150 mm, slant medium volume 5 mL, be unified in 28 DEG C, cultivate 6 ~ 8 d under 400 nm illumination, after spore is produced in mycelia induction, in inclined-plane, add 5 mL sterilized waters, with sterilizing bamboo let gently scrape lawn prepare spore suspension as screening sample for subsequent use.
Mixed with the 200 mL PDA nutrient agars being cooled to 45 DEG C after sterilizing by spore suspension, pouring internal diameter into by the amount of 20 mL/ plates is in the sterilizing flat board of 9 cm, is prepared into the plate that carries disease germs after naturally cooling.Mycelial growth suppresses method to adopt conventional cylinder plate method, each plate that carries disease germs is placed sterilizing Oxford cup, in cup, adds the above-mentioned screening sample of 200 microlitre with liquid-transfering gun.With 95% ethanol for negative control, often process repetition 3 times, 28 DEG C, cultivate 72 h under 400 nm illumination, measure antibacterial circle diameter by right-angled intersection method.
The compound method of above PDA substratum is (g/L): potato 200.0 g, sucrose 20.0 g, agar 20.0 g, tap water 1 L, pH nature.Peeling potatoes, is cut into block and boils 30 min, then use filtered through gauze, then sugaring, supplies water to 1 L after dissolving, 121 DEG C of sterilizing 30 min.
Experimental data shows: in all screening samples, derives from ALS-045 bacterial strain bacterium slag and resin extraction using alcohol sample (M sample) shows excellent antagonism botrytis cinerea energy for growth.Antibacterial circle diameter reaches 35 mm, 32 mm and 29 mm respectively, average 32 mm; Derive from ALS-045 strain culturing filtered liquid ethyl acetate and extract sample (Y sample) then without bacteriostatic activity.
Experimental data shows: Amberlite XAD-16 macroporous adsorbent resin is applicable to the subsequent disposal of the nutrient solution processing ALS-045 bacterial strain.
Two, greenhouse pot culture confirmatory experiment
Investigate test sample strains A LS-045 culture prevention effect to vegetables and fruit grey mold disease under greenhouse pot culture condition.Contrast medicament is: 50% iprodione wettable powder, is produced by Jilin Lik-Sang agrochemical agricultural chemicals company limited.
The selection cucumber that after planting about 10 d growing way is consistent, tomato, strawberry and grape cotyledon carry out provide protection and therapeutic action measures.
Provide protection: use cotton swab uniform application in cucumber, tomato, strawberry and grape cotyledon tow sides ALS-045 bacterial strain bacterium slag and resin ethanol test sample (M sample), after 24 h, cut blade, be wrapped on petiole with the cotton fully soaked through water.To cultivate the gray mold germ of about 7 d, break into the bacterium cake of diameter 5 mm with punch tool, bacterium faces down, and is positioned over the nearly blade tip of face of blade and nearly petiole place respectively places 1 piece.Often 4 repetitions are established in process, often repeat 4 leaves, and set clear water as blank.In the illumination box of 20 DEG C, 72 h(h illumination every day 12 are cultivated in moisturizing, and 12 h are dark).
Therapeutic action: will cultivate the gray mold germ of about 7 d, break into the bacterium cake of diameter 5 mm with punch tool, bacterium faces down, is positioned over the nearly blade tip of face of blade and nearly petiole place respectively places 1 piece.Often 4 repetitions are established in process, often repeat 4 leaves, and set clear water as blank.In the illumination box of 20 DEG C, 24 h(h illumination every day 12 are cultivated in moisturizing, 12 h are dark) after, by ALS-045 bacterial strain bacterium slag and resin ethanol test sample (M sample) cotton swab uniform application test plants cotyledon tow sides, blade is cut after liquid is dry, be wrapped on petiole with the cotton balls fully soaked through water, in the illumination box of 20 DEG C, moisturizing is cultivated.
Observe incidence: when blank is fully fallen ill, adopt cross method of masurement to measure each lesion diameter, carry out severity Scaling by Lesion size, by prevention effect formulae discovery.Grade scale is: 0 grade: without scab; 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: lesion diameter 20.1 more than mm.This prevention effect calculation formula is:
Disease index=
× 100
Prevention effect (%)=
× 100
Experimental result shows: ALS-045 bacterial strain bacterium slag and resin ethanol test sample (M sample) the greenhouse pot culture prevention effect to test plant disease good.Be 87.68% to compare with contrasting the average preventive effect of pharmaceutical treatment, except the average preventive effect of grape is except 55.25%, M sample is to the result for the treatment of of cucumber, tomato, Botrytis cinerea disease, and average preventive effect is all more than 70%.Wherein, being 72.23% for the average preventive effect of cucumber therapeutic, is 76.45% for the average preventive effect of tomato therapeutic, is 70.44% for the average preventive effect of strawberry therapeutic.The average preventive effect of contrast medicament and M sample is suitable.
Except, this M sample has good provide protection.Under protected mode model, for above-mentioned Four Plants, the average preventive effect of M sample reaches more than 80%.Wherein, be 82.14% for the average preventive effect of cucumber protectiveness, being 88.67% for the average preventive effect of tomato protectiveness, is 84.90% for the average preventive effect of strawberry preserve; be 80.43% for the average preventive effect of grape protectiveness, and to contrast the average preventive effect of medicament protectiveness be 84.24 %.
Above result display: the application prospect that ALS-045 bacterial strain is good.
three,the molecular biology identification of object strains A LS-045
1, extracting genome DNA, amplification and order-checking
By object bacterial strain
aLS-045be seeded in sterilized 50 mL ISP2 liquid nutrient mediums, 180 rpm, cultivate 3 d under 28 ° of C, carry out the extraction of genomic dna, amplification and order-checking.Wherein, the composition of ISP2 liquid nutrient medium is (g/L): yeast extract 4.0 g, malt extract 10.0 g, glucose 4.0 g, tap water 1 L, pH nature, prepare rear 121 ° of C sterilizing 20 min.
(1) genomic dna is extracted
Get appropriate 3-5 mL culture, 13000 rpm, centrifugal 1 min, abandons substratum.Add DNA extraction lysate 300 microlitre, vortex precipitates.65 DEG C of water-bath 30 min.Add isopyknic chloroform: primary isoamyl alcohol (24:1), vortex mixes, centrifugal 5 min of 13000 rpm.Get supernatant, add isopyknic Virahol, mixing, 13000 rpm, centrifugal 5 min.Abandon supernatant, add 70% ethanol mixing, centrifugal 1 min of 13000 rpm.Abandon ethanol, be inverted centrifuge tube, dry.Add 30 microlitre ddH
2o, dissolving DNA ,-20 DEG C of preservations.
(2) amplification of 16S rRNA gene and order-checking
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:
DNA profiling 1 microlitre (about 20 ~ 100 ng DNA);
10 × PCR buffered soln (100 mmol/L Tris-HCl, pH 8.3);
500 mmol/L KCl；
15 mmol/L MgCl
25 microlitres;
DNTPs mixture 4 microlitre (each dNTPs concentration is 2.5 mmol/L, TaKaRa);
1.25 U Taq enzyme (TaKaRa);
Two each 1 microlitres of primer (final concentration is 0.4 μm of ol/L);
Aseptic deionized water 50 microlitre.
Pcr amplification instrument is PE company of the GeneAmp PCR system(U.S.), amplification program is: 95 ° of C 4 min; 94 ° of C 1 min, 60 ° of C 1 min, 72 ° of C 1 min, 35 circulations; 72 ° of C 10 min.
PCR product 1% agarose gel electrophoresis detects, and then cuts out the fragment of 1500 bp sizes with scalpel, reclaims test kit (WATSON Gel Extraction Mini Kit) reclaim and purifying object fragment with glue.
(3) measure sequence with AB I PR ISM 377 – 96 sequencer, terminator is BigDye(Perkin-Elmer), sequencing primer is 27F and 1541R.
Sequencing reaction system:
Amplified production after purifying about 1 microlitre (about 30 ng),
BigDye 2.5 microlitre,
Primer 1 microlitre (about 3.2 pmol/L),
Aseptic deionized water 10 microlitre.
Amplification program is: 95 ° of C 1 min, 56 ° of C 1 min, 72 ° of C 1 min, 35 circulations; 72 ° of C, 5 min; 4 ° of C preserve.
By to ALS-045 bacterial strain 16S rRNA sequence through pcr amplification and order-checking, obtain length be 886 effective 16S rRNA gene base sequences.
2, the analysis of 16S rRNA gene base
The DNA sequence dna recorded is through NCBI(National Center for Biotechnology Information) obtain the 16S rRNA gene order of related species after BLAST engine search, arrange with ClustalX 1.8 software.Get rid of base deletion site, use adjacent method phylogenetic tree construction.Distance matrix calculates according to Kimura ' s two-parameter model,
bootstrap1000 sub-samplings are carried out in inspection.
Result shows: the position that object strains A LS-045 grows on tree at phyletic evolution reliable (
bootstrapvalue >70).Line segment scale (0.005) represents the branch length of 0.5% sequence difference.Grow on tree at phyletic evolution, strains A LS-045 and reference culture
streptomyces roseocinereus16S rRNA gene order maximum comparability reaches 99. 94%(and sees Fig. 1).
3. object strains A LS-045 sem observation
Spore suspension (method is with example 1) being prepared by ALS-045 bacterial strain, getting 200 microlitre even spread to preparing (ISP2 medium component is shown in example 1) on ISP2 agar plate.Sterilized cover glass is entered with 30 ~ 45° angle oblique cutting afterwards on flat board.Dull and stereotyped in 28 DEG C of thermostat containers, cultivate 15 d, treat media surface a large amount of have the mycelia of spore to spread to grow on cover glass and can stop cultivation.Carefully the cover glass being attached with mycelial growth is taken out with tweezers, be positioned in the vacant culture dish of 105 mm of sterilizing, at room temperature after fully dry 5 d, carry out mycelium surface metal spraying, can sem observation be carried out.The model of electron microscope is: Quanta 200(FEI company, the U.S.).
Streptomyces microorganism characteristic feature is presented: spore chain is longer is spirrillum, and spore is ellipsoid shape, and there is projection (see figure 2) on surface from sem observation result: strains A LS-045.Observe under natural light, this bacteria growing initial stage bacterium colony surface, in ivory buff, along with growth time extends, forms lark by whitewash redness gradually; Aerial hyphae is abundant, very thin; Substrate mycelium is not ruptured, for purple yellow; Produce the purple yellow pigment of solubility, there is the feature of typical chain mould.But the bacterium colony of this bacterium, mycelia color and pigment generate situation and reference culture
streptomyces roseocinereusthere are differences, still need and determine kind.
Comprehensive above 16S rRNA gene and microorganism characteristic feature, deduction strains A LS-045 belong to streptomyces (
streptomyces.sp) bacterial strain.
Four, the preparation of strains A LS-045 biotechnological formulation
Spore suspension (example 1) prepared by 50 ALS-045 bacterial strains, be inoculated in 100 L and be equipped with in the fermentor tank of 60 L sterilising liq ISP2 substratum (ISP2 substratum compound method is with example 3) that (fermentor tank model is GUJS-100.Zhenjiang Oriental Bio-engineering Technology Co., Ltd), 28 DEG C, 120 L/min air flows, 150 rpm cultivate 3 d, treat that having a large amount of mycelium pellet to grow in nutrient solution stops cultivating.With this nutrient solution for seed liquor, its culture transferring is equipped with to 500 L in the fermentor tank of 350 L sterilising liq improvement Gause I substratum (improvement Gause I substratum compound method is with example 1) that (fermentor tank model is GUJS-500.Zhenjiang Oriental Bio-engineering Technology Co., Ltd), 28 DEG C, 120 L/min air flows, 150 rpm cultivate 5 d, treat that having a large amount of mycelium pellet to grow in nutrient solution stops ventilation.
In culture system, sterilized Amberlite XAD-16 macroporous adsorbent resin is added, 28 DEG C, 200 rpm adsorption treatment 24 h in 5% ratio.Filtering fermentation liquor, filtrate abandons, and mycelium and resin-phase 90% industrial alcohol extract 3-5 time, and namely extracting solution concentrating under reduced pressure obtains this biotechnological formulation enriched material.500 L amplify fermentation and carry out twice altogether, and amount to and obtain crude extract 9274 g, its outward appearance is pale tan oil.
Be used for field plot bioorganism preparation by 1.5% proportional sampling preparation, solvent system volume ratio is:
Ethanol: Tween80: water=5:1:94
Enriched material is uniformly dispersed stable under this solvent systems.
Five, ALS-045 bacterial strain biotechnological formulation 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 envrionment conditions.
Test medicine is: 1.5% ALS-045 biotechnological formulation (being developed by institute of microbiology of Yunnan University).Contrast medicament is: the phonetic mould amine suspension agent of 400 g/L (Bayer Cropscience Co., Ltd's production) and 500 g/L iprodione suspending agents (Bayer Cropscience Co., Ltd's production).
Using method is spray method.Spraying equipment is 16L-8AH type knapsack electric sprayer.
Formulate corresponding spraying time according to each crop gray mold regularity of occurrence and development: at the beginning of disease, when namely sickness rate is no more than 5% generation, start first time dispenser, after this at interval of 5 ~ 7 d once, totally 3 times.Each reagent agent and clear water contrast are all undertaken by 750 L/ hectare consumptions.By the prevention effect of medicine inspecting institute of the Ministry of Agriculture " pesticide field efficacy medicine test criterion " drug effect method of calculation investigation crop.
In on February 6,7 days to 2014 January in 2014, carry out 1.5% ALS-045 biotechnological formulation control grey mould fruit rot of strawberry field control effectiveness test in United Town, Xishan District, Kunming City, Yunnan Province.Test-results shows: 1.5% ALS-045 biotechnological formulation (1800 g/ hectare dosage group) reaches 62.88% to the prevention effect of grey mould fruit rot of strawberry; The contrast phonetic mould amine suspension agent of medicament 400 g/L (750 g/ hectare) and the control μ μ μ effect of 500 g/L iprodione suspending agents (750 g/ hectare) to grey mould fruit rot of strawberry are respectively 81.79%, 78.97%.
In on September 1,2 days to 2014 August in 2014, carry out 1.5% ALS-045 biotechnological formulation control grape grey mould field control effectiveness test in Qujiang River town, Honghe state, Yunnan Province Jianshui County.Test-results shows: 1.5% ALS-045 biotechnological formulation (2700 g/ hectare dosage group) reaches 69.82% to the prevention effect of grape grey mould; The contrast phonetic mould amine suspension agent of medicament 400 g/L (750 g/ hectare) and 500 g/L iprodione suspending agents (750 g/ hectare) are respectively 82.48%, 79.86% to the prevention effect of grape grey mould.
On September 18,20 days to 2014 August in 2014, carried out 1.5% ALS-045 biotechnological formulation control gray mold of cucumber field control effectiveness test in Kunyang town, Jinning County, Kunming, Yunnan Province, test-results shows: 1.5% ALS-045 biotechnological formulation (1800 g/ hectare dosage group) is respectively 71.17% to the prevention effect of gray mold of cucumber; The contrast phonetic mould amine suspension agent (750g/ hectare) of medicament 400 g/L and 500 g/L iprodione suspending agents (750 g/ hectare) are respectively 81.44%, 80.15% to the prevention effect of gray mold of cucumber.
On November 9,10 days to 2014 October in 2014, carried out 1.5% ALS-045 biotechnological formulation control graw mold of tomato field control effectiveness test in Kunyang town, Jinning County, Kunming, Yunnan Province, test-results shows: 1.5% ALS-045 biotechnological formulation (1800 g/ hectare dosage group) reaches 63.69% to the prevention effect of graw mold of tomato; The contrast phonetic mould amine suspension agent of medicament 400 g/L (750 g/ hectare) and 500 g/L iprodione suspending agents (750 g/ hectare) are respectively 76.08%, 73.88% to the prevention effect of graw mold of tomato.
Above result shows: strains A LS-045 can be used as the excellent material of biotechnological formulation research and development, and its biotechnological formulation prepared has the excellent results of control vegetable and fruit grey mold disease, can be developed further into as green bio agricultural chemicals.
Claims (4)
1. a bacterial strain for anti-vegetable and fruit gray mold, is characterized in that: described bacterial strain is streptomycete bacterial strain
streptomyces.
spaLS-045, deposit number is CGMCCNo:10986.
2. prepare the method for the antagonist of bacterial strain as claimed in claim 1, comprise the following steps:
(1) spore suspension is prepared
The product spore actinomycetes deriving from ALS-045 bacterial strain are inoculated in and inoculate on the ISP2 agar slant of sterilizing, cultivate 4 ~ 7 d, formed after a large amount of spore until mycelium, in inclined-plane, add deionized water, gently scrape lawn with sterilizing bamboo let and prepare spore suspension for 28 DEG C;
Above-mentioned slant medium ISP2 substratum is: yeast extract 4.0 g, malt extract 10.0 g, glucose 4.0 g, agar 20.0 g, tap water 1 L;
(2) 50 ALS-045 bacterial strains are prepared spore suspension, be inoculated in 100 L and be equipped with in the fermentor tank of 60 L sterilising liq ISP2 substratum, 28 DEG C, 120 L/min air flows, 150 rpm cultivate 3 d, treat that namely have a large amount of mycelium pellet to grow in nutrient solution stops cultivating;
(3) with step (2) nutrient solution for seed liquor, culture transferring is equipped with in the fermentor tank of 350 L sterilising liq improvement Gause I substratum to 500 L, 28 DEG C, 120 L/min air flows, 150 rpm, cultivate 5 d, treat that namely have a large amount of mycelium pellet to grow in nutrient solution stops ventilation;
The compound method of aforesaid liquid improvement Gause I substratum is: W-Gum 10.0 g, sucrose 10.0 g, yeast extract 6.0 g, NaCl 0.5g, NaNO
32.0 g, K
2hPO
43H
2o 0.5 g, MgSO
47H
2o 0.5 g, FeSO
47H
2o 0.02 g, tap water 1 L, pH 7.4 ~ 7.6;
(4) in the culture system of step (3) fermentor tank, sterilized Amberlite XAD-16 macroporous adsorbent resin is added in 5% ratio, 28 DEG C, 200 rpm adsorption treatment 24 h, filtering fermentating liquid, abandon filtrate, mycelium and resin-phase 90% industrial alcohol extract 3 ~ 5 times, and namely extracting solution concentrating under reduced pressure obtains biotechnological formulation enriched material;
(5) circulation step (2) ~ (4), 500 L amplify fermentation twice, and amounting to acquisition outward appearance is brown yellow oil enriched material 9274 g, i.e. streptomycete bacterial strain Streptomyces. sp ALS-045.
3. the application of the antagonism vegetable and fruit gray mold of the antagonist of bacterial strain as claimed in claim 1.
4. application according to claim 3, it is characterized in that: get streptomycete bacterial strain Streptomyces. sp ALS-045 enriched material by 1.5% of solvent system volume ratio and add solvent system, the volume ratio of this solvent system is: ethanol: Tween80: water=5:1:94, and described ALS-045 enriched material is uniformly dispersed stable under this solvent systems.
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