CN1318642A - New streptomycete strain and its use - Google Patents
New streptomycete strain and its use Download PDFInfo
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- CN1318642A CN1318642A CN 00105993 CN00105993A CN1318642A CN 1318642 A CN1318642 A CN 1318642A CN 00105993 CN00105993 CN 00105993 CN 00105993 A CN00105993 A CN 00105993A CN 1318642 A CN1318642 A CN 1318642A
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- streptomycete
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- aleyrodid
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Abstract
The present invention relates to separated streptomyces orientalis strain Y31014 and streptomyces melanogenes strain Y31042-1 capable of controlling aleyrodids effectively. The present invention also relates to new biological pesticide and its use in preventing and controlling harmful creatures.
Description
The present invention relates to new east streptomycete (Streptomyces orientalis) and produce black streptomycete (Streptomyces melanogenes) bacterial strain, and uses thereof.
Have many kinds of insects can cause agricultural to go up heavy economic losses, and disseminate disease between plant, aleyrodid is one of the most flagrant Agricultural pests of the whole world.For example: in the time of 1981, tobacco aleyrodid (Bemisia tabaci) (Gennadius) is caused american cotton, cucurbit and 100,000,000 dollars of losses of lettuce.In the time of 1986, aleyrodid becomes the problem of Florida State, and the poinsettia that the tobacco aleyrodid makes the Florida State be worth 800 to 1,000 ten thousand dollars is subjected to about 200 ten thousand dollars loss.
Present known aleyrodid gnaws plants different more than 500 kinds.For example: sweet potato, tomato, beans, cotton, Radix Dauci Sativae, cassava, pumpkin class, lettuce, capsicum, eggplant, watermelon, and gherkin be the known host of this insect.Also known tobacco powder wind can be propagated the malicious disease that causes with microorganism of parasitosis more than 70 kinds.
In the time of 1991, Bemisia argentifolii (B.argentifolii Bellows and Perring) appears in Taiwan first.During nineteen ninety-five, Bemisia argentifolii causes the agricultural losses in 1000 hectares in Taiwan.
Aleyrodid is difficult to utilize known sterilant dispenser method control.There are many factors to cause leak in the insecticide control work.Have only a few sterilant commodity can effectively control aleyrodid.Yet a necessary week of these sterilants, thoroughly administered several times was just effective.In addition, most lifetime of aleyrodid is all stayed the leaf back side; Therefore, the grower's methodology that must adjust them just can improve the coverage of insecticide.The crack must make chemical spray agent can infiltrate the treetop and arrive the plant all surface by foot between the plant.
In addition, the chemical insecticide of inefficient use expends cost, and other remarkable shortcomings are arranged, and as environmental pollution, and peasant and human consumer is had potential health hazard.
Therefore need safer and more effective control aleyrodid method.Though year has been studied in biological control agent, still lifeless matter control agent commodity can successfully be prevented and treated aleyrodid at present.
USP 4,942,030 and USP 5,413,784 disclose and utilize fungi paecilomyces fumosoroseus (Paecilomyces fumosoroseus) and muscardine (Beauveria bassiana) control tobacco aleyrodid.
Streptomyces has been widely used in the manufacturing microbiotic, yet does not identify the streptomyces of the biological insecticidal activity with antagonism aleyrodid so far yet.
Now shockingly find the new bacterial strain of streptomyces, be called east streptomycete Y31014 and produce black streptomycete Y31042-1 that these bacterial strains have the activity of antagonism aleyrodid.
Therefore, one aspect of the present invention provides new bacterial strain east streptomycete Y31014 and produces black streptomycete Y31042-1.
Second aspect present invention provides the biopesticide that contains this new bacterial strain composition.
The present invention provides the method that contacts pest control via insect and new bacterial strain on the other hand.
Evaluation of microorganism and specificity analysis
New Y31014 and Y31042-1 bacterial strain are to separate in the geographic soil sample of Taiwan Province's Miaoshu.These microorganisms are identified through Xinzhu County ,Taiwan Province Foodstuff Industrial and Development Inst., are respectively the east streptomycete and produce black streptomycete.Its method and result are as follows:
Adopt international chain mould plan (International Streptomyces Project) (ISP) for analyzing methods analyst taxonomy and the morphological characteristic that the streptomyces characteristic is advised.
1. cell walls analysis
Get stem cell (10 milligrams) and insert in the test tube that contains 1 milliliter of 6N HCl, in 100 ℃ of following hydrolysis 18 hours.Hydrolyzed solution filters and is dry.Dry powder is dissolved in 0.4 ml distilled water, to the PTLC plate.Adopt methyl alcohol-H
2O-6N HCl-pyridine is as developing solution.After air-dry, use triketohydrindene hydrate then.Diaminopimelic acid (DAP) produces yellow-green colour, and other amino acid then produce red-purple.Referring to Ke Majiata people such as (Komagata), " lipid of cell divide and cell walls analytical method " (Lipid and Cell Wall Analysis in BacterialSystematics), Meth.Microbiol.19:161-207 (1987).
2. full cell glycan analysis method
Getting stem cell (50 milligrams) inserts and contains 1 milliliter of 1N H
2SO
4Test tube in, in 100 ℃ of following hydrolysis 2 hours.Utilize saturated Ba (OH)
2Adjust in the scope of hydrolyzed solution pH to 5.0-5.5.Hydrolyzed solution is centrifugal, collects upper strata suspension, and concentrates.Enriched material is dissolved in 0.4 ml distilled water, puts to filter paper then.Adopt N-methyl alcohol-H
2O-pyridine-toluene is as developing solution.Use the anilinephthalein acid esters and make the carbohydrate colour developing.6 carbon sugar produce brown in air-dry back, and 5 carbon sugar then produce pink.Referring to people such as Ke Majiata, " lipid of systematic bacteriology and cell walls analytical method ", Meth.Microbiol.19:161.207 (1987).
3. strain culturing specificity analysis
Cell is respectively at cultivating 14 days in yeast extract-wort agar (ISP#2 substratum), oatmeal agar (ISP#3 substratum), inorganic salt Starch Agar (ISP#4 substratum) and the glycerine-l-asparagine agar (ISP#5 substratum), with observation battalion's bacteria yarn quality, aerial hyphae quality, spore output and pigment production.Referring to uncommon woods people such as (Shiring), " characteristic analysis method of streptomyces species ", Int.J.Syst.Bacteriol., 16:313-340 (1966).
4. melanochrome produces analytical method
Get cell and in Tryptones-yeast extract nutritive medium (ISP#1 substratum), peptone-yeast extract agar (ISP#6 substratum) and tyrosine agar (ISP#7 substratum), cultivated respectively 7 and 14 days, observe melanochrome production situation.Referring to uncommon Lin Dengren, " characteristic analysis method of streptomyces species ", Int.J.Syst.Bacteriol., 16:313-340 (1966).
5. morphological specificity analysis
Downcut cell from ISP#2,3,4 and 5 substratum together with agar, in baking oven, dewater, on the ion coating machine, be coated with gold.Adopt sweep electron microscope (SEM) research form.
6. the utilization of carbohydrate and physiological property analysis
Cell culturing cell 7 and 14 days on the ISP#9 substratum that contains 1% sugar (D-glucose, L-arabinose, D-wood sugar, sucrose, D-fructose, rhamnosyl, raffinose, I-inositol, D-mannitol, Mierocrystalline cellulose, salicin), the observation of cell growth.Referring to uncommon Lin Dengren, " characteristic analysis method of streptomyces ", Int.J.Syst.Bacteriol., 16:313-340 (1966).
The results are shown in table 1 to 4.
The physiological property carbon source Y31014D-glucose of table 2.Y31014+
*D-wood sugar+D-fructose+sucrose-L-arabinose+rhamnosyl-raffinose+D-mannitol+I-inositol+Mierocrystalline cellulose-salicin+
*+: positive reaction ,-: negative reaction
The physiological property of table 4.Y31042-1
Carbon source Y31042-1
D-glucose+
*
The D-wood sugar+
D-fructose+
Sucrose-
L-Arab+
Rhamnosyl-
Raffinose+
The D-mannitol+
The I-inositol+
Mierocrystalline cellulose-
Salicin-
*+: positive reaction ,-: the cell walls amino acid of negative reaction cell walls amino acid and full cell carbohydrate analytical method Y31014 and two kinds of bacterial classifications of Y31042-1 and the sugared content of full cell are respectively LL-DAP and glucose, ribose.According to the strong people's " chemotaxonomy of ray fungi " (The Chemotaxonomy ofActinomycetes) such as (Lechevalier) of Lee's Shiva, A. Di now " actinomycete taxonomy " SIM of (Dieiz) and D.W. Tai Er (Thayer) (editor) announce No.6 especially, the explanation classification of USA, these bacterial classifications belong to chemotype IC, are classified as streptomyces.
Cultivar identification
(the international periodical (International Journal of systematic Bacteriology) of Actinobase and systematic bacteriology) shows that Y31014 is the most relevant with the east streptomycete when comparing with the standard strain isolated of bacterial classification, and Y31042-1 is the most relevant with the black streptomycete of product.
The preservation data
The black streptomycete Y31042-1 culture of east streptomycete Y31014 and product is according to budapest treaty, be deposited in American type culture collection (ATCC on August 20th, 1999,10801 University Boulevard, Manassas, VA 20110-2209, USA), preserving number is respectively ATCC PTA-558 and PTA-557.
The invention provides east streptomycete Y31014 and produce the biological pure growth of deceiving streptomycete Y31042-1.
Prior art is known can to obtain the microbial mutation kind not changing under its characteristic.For example: mutant can derive from the processing of chemistry or physical property mutagens, as: UV light, X-ray, gamma-radiation and chemical substance are as N-methyl-N '-nitro-N-nitrosoguanidine.The also known culture that for example screens mother strains that utilizes of prior art obtains the natural variation strain.Therefore, the present invention also relates to east streptomycete Y31014 and produces mutant strain or the variant that still keeps this strain characteristic among the black streptomycete Y31042-1.
New east streptomycete Y31014 and produce that black streptomycete Y31042-1 is single for known first aleyrodid is had highly toxic streptomycete from strain.
The black strepto-Y31042-1 of east streptomycete Y31014 and product can adhere to and infiltrate subsequently the crust of insect host.After infiltrating the target pest crust, the mattress filament begins to enter host tissue, and dying insect then overgrows with mycelium.Spore produces at host's outside surface.These spores can disperse and infect new host insect.
The black streptomycete Y31042-1 effect of east streptomycete Y31014 and product is rapid, is using in 3 to 7 days, and the effect of killing four nymph stage in age Bemisia argentifoliis reaches significant 100%.When the nutrient solution thinning ratio reaches 10
-3The time, can kill high to 70% insect.Spendable concentration is every ml of carrier about 7 * 10
9To 7 * 10
6CFU.
According to the purpose of using, the composition that the nutrient solution of fermentation can directly use or be deployed into suitable sprinkling, atomizing, dusting, broadcast sowing or pour into a mould uses.For example: composition can be formulated into suspension or the even liquid that looses, emulsion, the even diffusing liquid of oil, paste, fine powder or the granula of the ready-made solution that sprays, wettable powder, suspension, highly enriched water-based, oiliness or other character.When using, the nutrient solution and the present composition can directly connect and be applied to insect, leaf portion or its surrounding environment.
During preparation biopesticide composition, can get rid of moisture content, obtain the pure growth of microorganism by growth medium.Composition can prepare according to known way, for example: replenish active ingredient with acceptable carrier, assistant agent or thinner on the agricultural, as: can not suppress the emulsifying agent of microorganism growth, even powder or tensio-active agent.
Be applicable to that carrier of the present invention includes, but is not limited to: natural or synthetic mineral abrasive dust, for example: calcite, talcum, diatomite, polynite, atlapulgite, or the like.In order to improve the physical properties of composition, can add the silicate of polymolecularity or the absorbable polymer of polymolecularity.
Be applicable to that emulsifying agent of the present invention comprises (but being not limited to): nonionic and anionic emulsifier, for example: polyoxyethylene aliphatic alcohol ether, alkyl sulfonic ester, aromatic yl sulphonate, or the like.
Be applicable to that even powder of the present invention comprises (but being not limited to): xylogen-sulfite waste lye and methylcellulose gum, or the like.
Suitable tensio-active agent comprises (but being not limited to): be disclosed in the sanitising agent of MacKenzie and emulsifying agent handbook (McCutcheon ' s Detergents and Emul sifiers Annual, MC publishing company, Glen Rock, New Jersey, 1988); And tensio-active agent encyclopedia (Encyclopedia ofSurfactants), Vol, I-III (Chemical publishing company, New York, 1980-1981) the known surface promoting agent in.
Biopesticide composition of the present invention is at aleyrodid Bemisia, therefore can be used for the toxinosis of preventing and treating and preventing to be propagated by for example aphid, leafhopper and aleyrodid.
Fermentation culture of the present invention also can with powder or particulate vector combined administration.When sprinkling was used, powder or particle composite can be applied directly to insect, leaf portion or surrounding environment.But in order to prepare the microorganism pure growth of mixed powder or particulate vector, can get rid of the moisture content of growth medium, with any other particle that does not suppress microorganism growth or powdered material combination.Though microorganism can with the particulate vector combined administration, if during the nutrient solution uniform mixing of carrier and fermentation, can more conveniently and more even use.Biopesticide composition of the present invention makes the mixture of using comprise microbial spore and mycelium and carrier.The two the existence of spore and mycelium can promote to be clustered on the targeted insect.
Embodiment 1
The screening of bacterial classification Y31014 and Y31042-1
Earlier the microorganism that preserves is moved on the PDA flat board, cultivated 7 days down in 30 ℃.On the PDA flat board, take out 1/10th microorganism, be seeded in 500 milliliters of round-bottomed flasks that contain 100 milliliters of NGY substratum (NB8 gram, glucose 10 grams, yeast extract 5 grams, 1 liter in water).Microorganism is in 30 ℃, and 200rpm cultivated 24 hours down.Get 5 milliliters of cultures and move in another 500 milliliters of round-bottomed flasks that contain 100 milliliters of SSM331 (W-Gum 30 grams, soya bean protein 30 grams, molasses 10 grams, 1 liter in water) substratum, cultivated 72 hours.The culture dilution also prepares to be used for the active test of selecting.
Place the Bemisia argentifolii in two four nymph stages in age of row on slide glass, each row is contained 5 larvas, 1 centimeter at interval.Getting 3 microlitres drops on each larva according to the culture that respectively dilutes of above-mentioned preparation.Slide glass is inserted in 9 centimeters culture dish that contain 1 ml water filter membrane.After 3 to 7 days, with the microscopic examination result.
Find that in the time of preceding 3 days mortality ratio is not high.After 7 days, under the 10-2 thinning ratio, the larval mortality of handling through Y31014 and Y3104-1 bacterial strain is 100%.Be subjected to the Y31014 bacterium to infect the white group of discovery on the dead aleyrodid health.Be subjected to the polypide of Y31042-1 strain infection only to find a few group, but whole larva transmitted sorrel.
Embodiment 2
The activity research of Y31014 and Y31042-1 bacterial strain
Whether in order to measure the active substance (group) that causes aleyrodid death is the antibiotic substance that Y31014 and Y31042-1 bacterial strain produce, or is due to this microorganism itself, carries out following experiment.
Get Y31014 and Y31042-1 bacterium culture under 15000 rpm centrifugal 15 minutes, separate collection supernatant liquor and precipitation piece are for further test.In addition, also prepare Y31041 and Y31042-1 bacterial strain through the sterilization culture, or with the culture of acetone or ethyl acetate extraction.The method of these samples according to example 2 explanations is applied on the aleyrodid.The results are shown in table 5 and 6.
Table 5Y31014 bacterial classification mortality ratio (%)
*250 * 500 * 750 * 1000 * nutrient solution, 90 60 50 50 supernatants, 50 60 50 50 precipitation pieces 90 70 70 70 are through sterilization culture 0000 acetone extracts 0000 ethyl acetate extracts 0000*Thinning ratio
Table 6Y31042-1 bacterial classification mortality ratio (%)
*100 * 250 * 500 * nutrient solution, 60 50 40 supernatant liquors, 000 precipitation pieces 70 70 10 are through sterilization culture 000 acetone extracts 000 ethyl acetate extracts 000
*Thinning ratio
By table 6 as seen, the biological activity of Y31042-1 bacterial strain only appears in nutrient solution and the precipitation piece.According to table 5, biological activity is present in the supernatant liquor of Y31014 bacterial strain, yet its activity is lower, and finds white group on corpse.Therefore think that the active substance of Y31014 and Y31042-1 strain culture is a microorganism itself.The biological activity of Y31014 bacterium supernatant liquor mainly is to be derived to utilize the complete isolating spore of centrifuging.
Claims (6)
1. the biological pure growth of a microorganism east streptomycete (Streptomyces orientalis) Y31014, or its mutant strain or variant.
2. microorganism as claimed in claim 1, its preserving number are ATCC PTA-558.
3. the biological pure growth of black streptomycete (Streptomyces melanogenes) Y31042-1 of a production by biological, or its mutant strain or variant.
4. microorganism as claimed in claim 3, its preserving number are ATCC PTA-557.
5. biotic pesticide composition, it comprises at least a of significant quantity and goes up acceptable carrier according to each biological pure growth and agricultural in the claim 1 to 4.
6. the method for a prevention target insect, it comprises use significant quantity at least a as each biological pure growth in the claim 1 to 4.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00105993 CN1318642A (en) | 2000-04-18 | 2000-04-18 | New streptomycete strain and its use |
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|---|---|---|---|
| CN 00105993 CN1318642A (en) | 2000-04-18 | 2000-04-18 | New streptomycete strain and its use |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100369548C (en) * | 2006-09-21 | 2008-02-20 | 广东省农业科学院植物保护研究所 | Preparation method for insecticidal compound made from ocean microorganism and application thereof |
| CN100448984C (en) * | 2006-03-07 | 2009-01-07 | 广东省微生物研究所 | Streptomyces vietnamensis |
| CN101328468B (en) * | 2007-06-21 | 2012-05-23 | 东宇生物科技股份有限公司 | Antiallergic lactic acid bacteria |
| CN113005048A (en) * | 2020-12-14 | 2021-06-22 | 河南农业大学 | Streptomyces nigricans CYS22, metabolite thereof and application thereof |
-
2000
- 2000-04-18 CN CN 00105993 patent/CN1318642A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100448984C (en) * | 2006-03-07 | 2009-01-07 | 广东省微生物研究所 | Streptomyces vietnamensis |
| CN100369548C (en) * | 2006-09-21 | 2008-02-20 | 广东省农业科学院植物保护研究所 | Preparation method for insecticidal compound made from ocean microorganism and application thereof |
| CN101328468B (en) * | 2007-06-21 | 2012-05-23 | 东宇生物科技股份有限公司 | Antiallergic lactic acid bacteria |
| CN113005048A (en) * | 2020-12-14 | 2021-06-22 | 河南农业大学 | Streptomyces nigricans CYS22, metabolite thereof and application thereof |
| CN113005048B (en) * | 2020-12-14 | 2022-07-15 | 河南农业大学 | Streptomyces nigricans CYS22, metabolite thereof and application thereof |
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