CN101223879B - Method of preparing microorganisms pesticide for preventing vegetable fungi disease - Google Patents
Method of preparing microorganisms pesticide for preventing vegetable fungi disease Download PDFInfo
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- CN101223879B CN101223879B CN2007101568341A CN200710156834A CN101223879B CN 101223879 B CN101223879 B CN 101223879B CN 2007101568341 A CN2007101568341 A CN 2007101568341A CN 200710156834 A CN200710156834 A CN 200710156834A CN 101223879 B CN101223879 B CN 101223879B
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- pesticide
- streptomyces diastatochromogenes
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- MHVBAAGXKRZGHP-UHFFFAOYSA-N CNC(C(O)=O)C(C(N1)=O)=CNC1=O Chemical compound CNC(C(O)=O)C(C(N1)=O)=CNC1=O MHVBAAGXKRZGHP-UHFFFAOYSA-N 0.000 description 1
Abstract
The invention discloses a preparation method of a microbial pesticide for preventing fungal diseases of vegetables, and pertains to the technical field of microorganisms. The method adopts amylase chromogenic streptomyces D which is isolated and screened from a soil sample collected from Tianmu mountain in Lin'an, Hangzhou of Zhejiang Province to be treated with such procedures as bacteria activation culture, fermentation culture, extraction of ferment substance, provision of agricultural auxiliary pesticide to a refined active compound, etc. to be made into agricultural drug with a plurality of dosage forms. The microbial pesticide can be used for preventing fungal diseases of vegetables such as cucumber fusarium wilt, tomato gray mold, etc., and has better and safer control effect than the conventional chemical agent.
Description
Technical field
The present invention relates to microbial technology field, particularly relate to the biological and ecological methods to prevent plant disease, pests, and erosion actinomycetes---the cultivation of streptomyces diastatochromogenes D and the separation of metabolite and application.
Background technology
Actinomycetes (Actinomycetes) are very close with human relation.According to statistics, in the antibiotic of having found, have 61.7% to be produced by actinomycetes.Antibiotic commonly used is except that penicillin and cephalosporin class, and the overwhelming majority is actinomycetic product.Soil is the important place of actinomycetes perch, and it is to develop new medicine and the requisite element task of novel pesticide in the present world wide that the actinomycetes that obtain to have the high-efficiency antimicrobial activity from soil produce antibiotic.
At present, mainly adopt chemical pesticide to kill pathogen, but chemical pesticide still is difficult to effective solution so far to people and animals' toxic and side effect and residue problem to the control of plant disease.Utilize the active substance of the disease-resistant fungal pathogens that actinomycetes produce to carry out the biological control of plant pathogenic fungi disease, then have the cycle short, be easy to research, advantage such as be convenient to produce, nontoxic.
The present invention is separated to strain biological and ecological methods to prevent plant disease, pests, and erosion actinomycetes from the soil in Hangzhou, and the antimicrobial component in this biological and ecological methods to prevent plant disease, pests, and erosion actinomycete fermentation metabolite is separated, and has obtained main active, through four big spectral analysis, and clear and definite its structural formula.This invention provides good starting strain and bioactive compound for the further development and the exploitation of microbial pesticide.
Summary of the invention
The objective of the invention is the dangerous and less deficiency of microbial pesticide kind, a kind of actinomycetes that separate from the soil of Hangzhou-streptomyces diastatochromogenes D is provided at chemical pesticide; Another object of the present invention provides utilizes this bacterial strain to be prepared into microbial pesticide of preventing and treating the part fungal diseases of plants and preparation method thereof by its metabolite of fermented extracted.
The separation of streptomyces diastatochromogenes of the present invention, screening and evaluation:
Be in August, 2006 in the soil sample that gather in the Tian Mu Shan Mountain, Zhejiang Hangzhou Linan, obtain and preserve through separation, cultivation, fermentation, determination of activity and to the steps such as screening that suppress fungal diseases of plants pathogen effective strain; Be accredited as streptomyces diastatochromogenes (Streptomycesdiastatochromogenes) through microbial taxonomy, name and be streptomyces diastatochromogenes (Streptomycesdiastatochromogenes) D.This bacterial strain is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preservation date on May 25th, 2007, preservation registration number CGMCC No.2060.
Streptomyces diastatochromogenes D microscopic morphology feature: straight, hook-shaped, the loose spirality of fibrillae of spores, spore is oval, ellipse or oval.
Streptomyces diastatochromogenes D of the present invention, can obtain the inhibited reactive compound [2 of part fungal diseases of plants pathogen through fermented extracted, 4-Dioxo-1-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yl)-1,2,3,4-tetrahydro-pyrimidin-5-yl]-methylamino-acetic acid.
The object of the invention is achieved through the following technical solutions:
A kind of preparation method who prevents and treats fungal diseases of plants microbial pesticides such as graw mold of tomato and cucumber fusarium axysporum, carry out according to the following steps:
(1) activation culture of bacterial classification: will separate the streptomyces diastatochromogenes D bacterial classification of preserving and move in vitro on the Gause I inclined-plane, and under 28 ℃ ± 1 ℃ condition, be cultured to the test tube slant and cover with spore;
(2) fermented and cultured: fermentation culture is to contain soybean cake powder 20g among every 1000mL, corn flour 15g, wheat bran 10g, NH
4NO
33g, (NH
4)
2SO
45g, CaCO
35g, MgSO
41g, sterilization, to be cooled to 40 ℃, inoculation streptomyces diastatochromogenes D under 28 ℃ ± 1 ℃ condition, cultivated 5 days to fermentation culture under aseptic condition;
(3) fermentation product extracts: fermentation finishes, and collects zymotic fluid, chemical method extraction routinely, chromatography, and separation and purification obtains reactive compound
[2,4-Dioxo-1-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yl)-1,2,3,4-tetrahydro-pyrimidin-5-yl]-methylamino-acetic acid, this compound molecule formula is C
13H
19O
9N
3, structural formula is formula as follows;
(4) this reactive compound is equipped with the agricultural drugs auxiliary material, is prepared into the agricultural drugs of multiple formulation.
Beneficial effect of the present invention:
The one, the present invention separates metabolite output height and part fungal diseases of plants pathogen is had stronger inhibiting streptomyces diastatochromogenes D from Hangzhou soil, and this provides a kind of microbial strains for control fungal diseases of plants pathogen on the agricultural;
The 2nd, the invention provides and from the fermentation product of streptomyces diastatochromogenes D, extract its active metabolite [2,4-Dioxo-1-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yl)-1,2,3,4-tetrahydro-pyrimidin-5-yl]-methylamino-acetic acid, and and then be prepared into the preparation method of the microbial pesticide that can prevent and treat fungal diseases of plants; This agricultural chemicals is applied to the control of fungal diseases of plants, and its effect is than conventional chemical medicament slightly excellent (seeing the routine table 1 of test), for the exploitation of disinfectant use in agriculture has increased new approach.
Embodiment
Separation and the screening of embodiment 1:(streptomyces diastatochromogenes D)
The present invention in August, 2006 in the soil sample that gather in the Tian Mu Shan Mountain, Linan, Zhejiang, through separation, cultivation, fermentation, determination of activity and to suppressing the steps such as screening of fungal diseases of plants pathogen effective strain, therefrom obtain streptomyces diastatochromogenes (Streptomyces diastatochromogenes) D, preserve.
Streptomyces diastatochromogenes (Streptomyces diastatochromogenes) D bacteriostatic activity is measured: get its fermented supernatant fluid 1mL in sterile petri dish, be cooled to 50 ℃ the rapid mixing of PDA medium with 9mL, cucumber fusarium axysporum that it is 4mm that the cooling back is put 1 diameter respectively on each medium plane or graw mold of tomato etc. are for examination bacterium bacterium cake, the bacterium cake is connected to culture dish central authorities (the culture dish diameter is 9cm), put 28 ℃ of cultivation 36h in the incubator, handle in contrast with sterile water, repeat 3 times; Adopt the right-angled intersection method to measure colony diameter, calculate inhibiting rate with following formula:
Measurement result shows: streptomyces diastatochromogenes (Streptomyces diastatochromogenes) D metabolite is respectively 83.8% and 78% to the inhibiting rate of cucumber fusarium axysporum and botrytis cinerea.
The preparation method of embodiment 2:(streptomyces diastatochromogenes D fermentating metabolism product)
Carry out according to the following steps:
(1) bacterial classification test tube slant activation culture: slant medium is the Gause I medium, and its component and content are: K
2HPO
40.5g, KNO
31g, MgSO
40.5g, soluble starch 20g, NaCl 0.5g, FeSO
40.01g, agar 20g, supply water to 1L after dissolving; With the spore of a little streptomyces diastatochromogenes D of oese picking, (in 15 * 150mm), put interior 28 ℃ ± 1 ℃ of incubator, activation culture 96h covers with spore to the test tube slant to sterilized Gause I medium test tube under aseptic condition;
(2) fermented and cultured: fermentation culture is to contain soybean cake powder 20g among every 1000mL, corn flour 15g, wheat bran 10g, NH
4NO
33g, (NH
4)
2SO
45g, CaCO
35g, MgSO
41g, surplus is a water; The bottled 40mL fermentation culture of every 300mL triangle, prepare back 121 ℃ the sterilization 20min, to be cooled to 40 ℃ under aseptic condition, picking one platinum ring streptomyces diastatochromogenes D spore inoculating, 28 ℃ ± 1 ℃, fermented and cultured 5 days;
(3) fermentation product extracts: collect zymotic fluid after fermentation finishes, under vacuum condition, be concentrated into medicinal extract, dissolve with isopropyl alcohol again, the centrifuging and taking supernatant, be concentrated into medicinal extract, then go up silica gel column chromatography, with isopropyl alcohol and water (volume ratio 10%～90%) wash-out, separation and purification gets reactive compound, and [2,4-Dioxo-1-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yl)-1,2,3,4-tetrahydro-pyrimidin-5-yl]-methylamino-acetic acid.
Streptomyces diastatochromogenes D metabolite described in following examples 3,4 is embodiment 2 products made therebies; The percentage composition of described product is mass percent.
Embodiment 3:(utilizes streptomyces diastatochromogenes D fermentating metabolism product to be prepared into wetting powder)
Wherein:
White carbon: (the rich strange chemical industry Co., Ltd in Shanghai produces)
Blue star auxiliary reagent factory, farming newborn 0203-B:(Xingtai produces)
Sodium lignin sulfonate: (the Shanghai prestige is chemical industry Co., Ltd and produces)
Precipitated calcium carbonate: (Shanghai DaYu biochemistry Co., Ltd's production)
Product contains 1.5% reactive compound [2,4-Dioxo-1-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yl)-1,2,3,4-tetrahydro-pyrimidin-5-yl]-methylamino-acetic acid, the white carbon of adding 2% is as adsorbent, and add 1% agricultural newborn 0203-B and 3% sodium lignin sulfonate auxiliary material, supply 100% with precipitated calcium carbonate at last, behind the mixing, make wetting powder (claiming " product A " in the following content) through comminution by gas stream.
Embodiment 4:(utilizes streptomyces diastatochromogenes D fermentating metabolism product to be prepared into missible oil)
Wherein:
Ethanol (production of Shanghai Shen Xing pharmaceutical factory)
0204 (production of Xingtai blue star auxiliary reagent factory) of farming breast
Dimethylbenzene (production of Shanghai Shen Xing pharmaceutical factory)
The reactive compound [2 of product 2%, 4-Dioxo-1-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yl)-1,2,3,4-tetrahydro-pyrimidin-5-yl]-methylamino-acetic acid, the dissolve with ethanol with 4% adds quality 5% agricultural newborn 0204-C as emulsifier, supply 100% with dimethylbenzene at last, stirring and evenly mixing is made missible oil (claiming " product B " in the following content).
Test example: (product of the present invention and conventional pesticide control efficiency comparative trial)
" product A, B " prevented and treated test to plant diseases such as cucumber fusarium axysporum or graw mold of tomato, is contrast with conventional chemical agricultural chemicals, clear water, and shown in table 1, table 2, the disinfectant use in agriculture of trial-production " product A, B " is slightly more excellent than conventional chemical pesticide control effect.
Table 1 product A is to graw mold of tomato preventive effect statistical form
(test of pesticide effectiveness is all carried out in test farmland, suburbs, Hangzhou in July, 2007-September)
| Product and contrast medicine | Extension rate | Using method | Proofread and correct preventive effect (%) |
| Graw mold of tomato | ? | ? | ? |
| Product A | 1000? | Spraying | 90.9? |
| 1500? | Spraying | 88.5? | ? |
| 2000? | Spraying | 85.1? | ? |
| But 70% thiophanate methyl wet-milling | 1000? | Spraying | 90.7? |
| Clear water | -? | Spraying | 4.3? |
Table 2 product B is to cucumber fusarium axysporum preventive effect statistical form
(test of pesticide effectiveness is all carried out in test farmland, suburbs, Hangzhou in July, 2007-September)
| Product and contrast medicine | Extension rate | Using method | Proofread and correct preventive effect (%) |
| Cucumber fusarium axysporum | ? | ? | ? |
| Product B | 800? | Spraying | 93.8? |
| 1200? | Spraying | 85.3? | ? |
| 1600? | Spraying | 79.0? | ? |
| But 50% carbendazim wet-milling | 500? | Spraying | 84.4? |
| Clear water | -? | Spraying | -10.2? |
Claims (1)
1. preparation method who prevents and treats the microbial pesticide of cucumber fusarium axysporum and graw mold of tomato may further comprise the steps:
(1) activation culture of bacterial classification: streptomyces diastatochromogenes (Streptomycesdiastatochromogenes) D bacterial classification is moved in vitro on the Gause I inclined-plane, under 28 ℃ ± 1 ℃ condition, be cultured to the test tube slant and cover with spore, described streptomyces diastatochromogenes D bacterial classification is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, and preservation registration number is CGMCC No.2060;
(2) fermented and cultured: fermentation culture is to contain soybean cake powder 20g among every 1000mL, corn flour 15g, wheat bran 10g, NH
4NO
33g, (NH
4)
2SO
45g, CaCO
35g, MgSO
41g, to be cooled to 40 ℃, inoculation streptomyces diastatochromogenes D under 28 ℃ ± 1 ℃ condition, cultivated 5 days to fermentation culture under aseptic condition;
(3) fermentation product extracts: fermentation finishes, and collects zymotic fluid, chemical method extraction routinely, chromatography, and separation and purification obtains reactive compound; The structural formula of this reactive compound is as follows:
(4) this compound is equipped with the agricultural drugs auxiliary material, is prepared into the agricultural drugs of multiple formulation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007101568341A CN101223879B (en) | 2007-11-19 | 2007-11-19 | Method of preparing microorganisms pesticide for preventing vegetable fungi disease |
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|---|---|---|---|
| CN2007101568341A CN101223879B (en) | 2007-11-19 | 2007-11-19 | Method of preparing microorganisms pesticide for preventing vegetable fungi disease |
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| Publication Number | Publication Date |
|---|---|
| CN101223879A CN101223879A (en) | 2008-07-23 |
| CN101223879B true CN101223879B (en) | 2011-05-11 |
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|---|---|---|---|
| CN2007101568341A Expired - Fee Related CN101223879B (en) | 2007-11-19 | 2007-11-19 | Method of preparing microorganisms pesticide for preventing vegetable fungi disease |
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101961014B (en) * | 2010-03-19 | 2013-04-24 | 中国计量学院 | Method for preparing microbial pesticide for preventing and controlling fungal diseases of vegetables |
| CN102217656B (en) * | 2011-04-08 | 2013-01-30 | 中国计量学院 | Application of alternaria alternata metabolite to controlling Botrytis cirerea |
| CN102210330B (en) * | 2011-04-08 | 2013-01-30 | 中国计量学院 | Application of metabolites of nigrospora oryzae 46 in prevention and treatment of botrytis cinerea |
| CN102204569B (en) * | 2011-04-08 | 2013-01-30 | 中国计量学院 | Application of Aspergillus restrictus 28 metabolic product in controlling rice thanatephorus cucumeris |
| CN102210329B (en) * | 2011-04-08 | 2013-01-30 | 中国计量学院 | Application of Aspergillus restrictus 28-generation product on preventing and curing cucumber Rhizoctonia solani |
| CN102210331B (en) * | 2011-04-08 | 2013-01-30 | 中国计量学院 | Application of Nigrospora oryzae 46 metabolic product on preventing and treating Rhizoctonia solani |
| CN112640918B (en) * | 2020-12-14 | 2021-08-17 | 中国计量大学 | Application of streptomyces diastatochromogenes metabolite in preventing and treating dendrobium officinale diseases |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4056615A (en) * | 1975-05-28 | 1977-11-01 | U C B Societe Anonyme | Lucknomycin and process for producing same |
| CN1286025A (en) * | 2000-08-14 | 2001-03-07 | 浙江省农业科学院微生物研究所 | Duokangling as antibiotic-type agricultural chemical and its preparing process |
-
2007
- 2007-11-19 CN CN2007101568341A patent/CN101223879B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4056615A (en) * | 1975-05-28 | 1977-11-01 | U C B Societe Anonyme | Lucknomycin and process for producing same |
| CN1286025A (en) * | 2000-08-14 | 2001-03-07 | 浙江省农业科学院微生物研究所 | Duokangling as antibiotic-type agricultural chemical and its preparing process |
Non-Patent Citations (4)
| Title |
|---|
| 孟晓峰 等.微生物来源的免疫抑制剂的研究进展.国外医药抗生素分册18 6.1997,18(6),401-408，417. |
| 孟晓峰 等.微生物来源的免疫抑制剂的研究进展.国外医药抗生素分册18 6.1997,18(6),401-408,417. * |
| 石孟芝.微生物杀灭钉螺研究概况.实用预防医学10 3.2003,10(3),435-437. |
| 石孟芝.微生物杀灭钉螺研究概况.实用预防医学10 3.2003,10(3),435-437. * |
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|---|---|
| CN101223879A (en) | 2008-07-23 |
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