CN101961014B - Method for preparing microbial pesticide for preventing and controlling fungal diseases of vegetables - Google Patents
Method for preparing microbial pesticide for preventing and controlling fungal diseases of vegetables Download PDFInfo
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- CN101961014B CN101961014B CN 201010127364 CN201010127364A CN101961014B CN 101961014 B CN101961014 B CN 101961014B CN 201010127364 CN201010127364 CN 201010127364 CN 201010127364 A CN201010127364 A CN 201010127364A CN 101961014 B CN101961014 B CN 101961014B
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Abstract
The invention discloses a method for preparing a microbial pesticide for preventing and controlling fungal diseases of vegetables, and belongs to the technical field of microorganisms. In the method, streptomyces diastatochromogenes D which is separated and screened from a soil sample collected from Tianmu mountain of Lin'an county of Hangzhou city of Zhejiang province is utilized; and the agricultural pesticide of various formulations is prepared by active culturing of strain, fermentation culturing, leavening extraction and combination of an extracted active compound and auxiliary materials of the agricultural pesticide and the like. The microbial pesticide can be used for preventing and controlling the fungal disease of the vegetables, such as cucumber fusarium wilt, tomato gray mold or tomato early blight or the like; and the prevention and control effect of the pesticide is equivalent or slightly prior to that of the conventional chemical agent and the pesticide is safe.
Description
Technical field
The present invention relates to microbial technology field, particularly relate to biocontrol actinomycetes---streptomyces diastatochromogenes (Streptomyces diastatochromogenes) cultivation of 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 70% to be about actinomycetes and to produce.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.
To the control of plant disease, mainly adopt the method for chemical pesticide control at present.Although chemical pesticide has comparatively ideal control efficiency to multiple diseases, long-term use can be brought out the pesticide resistance of germ.In addition, the frequent use of agricultural chemicals has not only increased the production cost of agricultural product, and Pesticide Residue is also very serious.Utilize the active substance of the disease-resistant fungal pathogens that actinomycetes produce to carry out the biological control of fungal diseases of plants, then have the cycle short, be easy to research, the advantage such as be convenient to produce, nontoxic.
The report of toyokamycin activity mainly concentrated on anti-tumor aspect in the past, but because its toxicity to human normal cell line is too large, can't be applied to clinical.Aspect the report of antibacterial activity, toyokamycin mainly suppresses candida albicans, new Cryptococcus, mycobacterium graminis and staphylococcus aureus etc. be not almost to phytopathogen (except the Pyricularia oryzae (Magnaporthe grisea)) inhibiting relevant report (Chen Yi, Li Zhou, Wei Yuzhen, Deng. the research of the nucleoside antibiotic 3431 that rhizospheric actinomyces I06-03431 produces. biology magazine, 2009,26 (4): 17-20).This research is by finding the research of streptomyces diastatochromogenes Antibacterial Constituents toyokamycin, toyokamycin low concentration just can strong inhibition cucumber fusarium axysporum (FusariumOxysporum), botrytis cinerea (Botrytis Cinerea) and tomato early blight bacterium (Alternaria solani) spore germination and mycelial growth.The present invention has filled up this blank just, has widened the application of toyokamycin.Streptomyces diastatochromogenes produces the fermentation level of toyokamycin at 105.6mg/L among simultaneously the present invention, compare document (M.C.Flickinger, M.Greenstein, C.Bremmon, et al.Strain selection, medium development and scale-up of toyocamycin production byStreptomyces chrestomyceticus.Bioprocess Engineering, 5 (1990): the product element level of 143-153) reporting approximately 12mg/L will exceed 7.8 times.
Summary of the invention
The present invention is separated to a strain biocontrol actinomycetes from the soil in Hangzhou, be accredited as streptomyces diastatochromogenes (Streptomyces diastatochromogenes) through microbial taxonomy, and the antimicrobial component in this biocontrol actinomycetes fermentating metabolism product separated, obtained active component, through four large spectral analysis, prove known nucleoside antibiotic, toyokamycin (Toyocamycin).This invention provides good starting strain and bioactive compound for further development and the exploitation of microbial pesticide.
The objective of the invention is provides a kind of actinomycetes that separate-streptomyces diastatochromogenes D for the deficiency that chemical pesticide is dangerous and the microbial pesticide kind is less from the soil of Hangzhou; Another object of the present invention provides microbial pesticide that utilizes this bacterial strain to be prepared into control Activities of Some Plants fungal disease by its metabolite of fermentation extraction and preparation method thereof.
The separation of streptomyces diastatochromogenes of the present invention, screening and evaluation:
Be in August, 2006 in the soil sample that the Tian Mu Shan Mountain, Zhejiang Hangzhou Linan gathers, 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 (Streptomyces diastatochromogenes) through microbial taxonomy, name and be streptomyces diastatochromogenes (Streptomyces diastatochromogenes) 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.The preservation file is referring to the open text of patent ZL200710156833.7, and publication number is CN101225368A.
Streptomyces diastatochromogenes D micro-morphology: fibrillae of spores is straight, hook-shaped, loose-screw shape, and spore is oval, ellipse or oval.
Streptomyces diastatochromogenes D of the present invention can obtain the inhibited reactive compound toyokamycin (Toyocamycin) of Activities of Some Plants fungal disease pathogen through the fermentation extraction.
The object of the invention is achieved through the following technical solutions:
A kind of preparation method who prevents and treats the fungal diseases of plants microbial pesticides such as graw mold of tomato, cucumber fusarium axysporum or early blight of tomato, 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 corn flour 21.5g among every 1000mL, soluble starch 10g, wheat bran 5g, CaCO
32.5g, NH
4NO
31.5g, FeSO
40.5g, KH
2PO
41.5g, 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: ferment complete, collect zymotic fluid, and routinely chemical method extraction, chromatography, separation and purification obtains reactive compound Toyocamycin, and this compound molecule formula is C
12H
13N
5O
4, structural formula is seen following formula;
(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, it is high and Activities of Some Plants fungal disease pathogen had stronger inhibiting streptomyces diastatochromogenes D that the present invention separates metabolite output 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 from the fermentation product of streptomyces diastatochromogenes D and to extract its active metabolite toyokamycin Toyocamycin, and and then be prepared into the preparation method of the microbial pesticide that can prevent and treat fungal diseases of plants; This pesticides application is in the control of fungal diseases of plants, and its effect similar or slightly excellent than the conventional chemical medicament (seeing test example table 1-3) is for the exploitation of disinfectant use in agriculture has increased new approach.
Embodiment
The isolation and screening of embodiment 1:(streptomyces diastatochromogenes D)
The present invention in August, 2006 in the soil sample that the Tian Mu Shan Mountain, Linan, Zhejiang gathers, 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 Antibacterial Activity: get its fermented supernatant fluid 1mL in sterile petri dish, be cooled to 50 ℃ the rapid mixing of PDA medium with 9mL, the cucumber fusarium axysporum that it is 4mm that cooling is put respectively 1 diameter on each medium plane afterwards or botrytis cinerea or tomato early blight bacterium 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, process 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 86.9%, 83% and 85.2% to the inhibiting rate of cucumber fusarium axysporum, botrytis cinerea and tomato early blight bacterium.
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, NaCl0.5g, 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 yellow corn powder 21.5g among every 1000mL, soluble starch 10g, wheat bran 5g, CaCO
32.5g, NH
4NO
31.5g, FeSO
40.5g, KH
2PO
41.5g surplus is water; The bottled 40mL fermentation culture of every 300mL triangle, prepare rear 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: complete rear collection zymotic fluid ferments, adding isopyknic n-butanol in the zymotic fluid extracts, get the upper strata, and under vacuum condition, be concentrated into medicinal extract, with dissolved in distilled water, the centrifuging and taking supernatant is then gone up Sephadex LH20 column chromatography again, with ethanol and the distilled water gradient elution of volume ratio 10%～90%, separation and purification gets reactive compound Toyocamycin.
Streptomyces diastatochromogenes produces the fermentation level of toyokamycin at 105.6mg/L among the present invention, compare document (M.C.Flickinger, M.Greenstein, C.Bremmon, et al.Strain selection, medium development and scale-upof toyocamycin production by Streptomyces chrestomyceticus.Bioprocess Engineering, 5 (1990): the product element level of 143-153) reporting approximately 12mg/L will exceed 7.8 times.
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 Toyocamycin, 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 " products A " in the following content) through air-flow crushing.
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 Toyocamycin of product 2%, the ethanol dissolving with 4% adds quality 5% agricultural newborn 0204-C as emulsifier, supplies 100% with dimethylbenzene at last, and 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)
" products A, B " prevented and treated test to plant diseases such as cucumber fusarium axysporum or graw mold of tomato or earlys blight of tomato, take conventional chemical agricultural chemicals, clear water as contrast, shown in table 1, table 2 and table 3, the disinfectant use in agriculture of trial-production " products A, B " is more similar or slightly excellent than conventional chemical pesticide control effect.
Table 1 products A is to graw mold of tomato preventive effect statistical form
(test of pesticide effectiveness is all carried out in Hangzhou test farmland in July, 2009-September)
Table 2 product B is to cucumber fusarium axysporum preventive effect statistical form
(test of pesticide effectiveness is all carried out in Hangzhou test farmland in July, 2009-September)
Table 3 product B is to early blight of tomato preventive effect statistical form
(test of pesticide effectiveness is all carried out in Hangzhou test farmland in July, 2009-September)
Claims (3)
1. preparation method who prevents and treats the microbial pesticide of cucumber fusarium axysporum, early blight of tomato or graw mold of tomato may further comprise the steps:
(1) activation culture of bacterial classification: streptomyces diastatochromogenes (Streptomyces diastatochromogenes) 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 corn flour 21.5g among every 1000mL, soluble starch 10g, wheat bran 5g, CaCO
32.5g, NH
4NO
31.5g, FeSO
40.5g, KH
2PO
41.5g 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: ferment complete, collect zymotic fluid, and routinely chemical method extraction, chromatography, separation and purification obtains the reactive compound toyokamycin; 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.
2. according to claim 1 method, wherein in the fermentation product of step (3) extracts, concrete steps are: the complete rear collection zymotic fluid that ferments, and add isopyknic n-butanol in the zymotic fluid and extract, get the upper strata, and under vacuum condition, be concentrated into medicinal extract, with dissolved in distilled water, the centrifuging and taking supernatant is then gone up Sephadex LH20 column chromatography again, with ethanol and the distilled water gradient elution of volume ratio 10%～90%, separation and purification gets reactive compound.
3. according to claim 1 preparation method wherein in step (4), is prepared into the agricultural drugs of wetting powder or missible oil.
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| CN 201010127364 CN101961014B (en) | 2010-03-19 | 2010-03-19 | Method for preparing microbial pesticide for preventing and controlling fungal diseases of vegetables |
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| CN101961014B true CN101961014B (en) | 2013-04-24 |
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| 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 |
| CN102217656B (en) * | 2011-04-08 | 2013-01-30 | 中国计量学院 | Application of alternaria alternata metabolite to controlling Botrytis cirerea |
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| CN102899356A (en) * | 2012-09-23 | 2013-01-30 | 江苏丘陵地区镇江农业科学研究所 | Application of Streptomyces corchorusii NF0919 strain metabolite in grape disease control |
| CN102911970A (en) * | 2012-09-23 | 2013-02-06 | 江苏丘陵地区镇江农业科学研究所 | Application of metabolic product of streptomyces corchorusii NF0919 bacterial strain to preventing and treating tomato diseases |
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| CN103651607B (en) * | 2013-12-18 | 2015-08-05 | 青岛百瑞吉生物工程有限公司 | A kind of vegetables biopesticide and preparation method thereof |
| CN103960288B (en) * | 2014-03-31 | 2015-10-14 | 中国计量学院 | The application of a kind of mixing formula preparation in control brown planthopper |
| CN104342388B (en) * | 2014-09-29 | 2016-11-02 | 中国计量学院 | Streptomycete bacterial strain and prevent and treat tomato wilt use in conjunction |
| CN105624046B (en) * | 2016-01-26 | 2019-03-12 | 华中农业大学 | It is a kind of to prevent and treat mycotic black fungus bacterial and its application |
| CN106635878A (en) * | 2016-10-25 | 2017-05-10 | 扬州大学 | Streptomyces sp. 5X4 and applications thereof as biocontrol bacterium in preventing and treating plant diseases |
| 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 (3)
| 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 |
| CN101223879A (en) * | 2007-11-19 | 2008-07-23 | 中国计量学院 | Method of preparing microorganisms pesticide for preventing vegetable fungi disease |
-
2010
- 2010-03-19 CN CN 201010127364 patent/CN101961014B/en not_active Expired - Fee Related
Patent Citations (3)
| 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 |
| CN101223879A (en) * | 2007-11-19 | 2008-07-23 | 中国计量学院 | Method of preparing microorganisms pesticide for preventing vegetable fungi disease |
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