CN103222478A - Use of Streptomyces sioyaensis metabolite in prevention and control of Fusarium oxyspirum f. sp. niveum - Google Patents
Use of Streptomyces sioyaensis metabolite in prevention and control of Fusarium oxyspirum f. sp. niveum Download PDFInfo
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- CN103222478A CN103222478A CN2013100080228A CN201310008022A CN103222478A CN 103222478 A CN103222478 A CN 103222478A CN 2013100080228 A CN2013100080228 A CN 2013100080228A CN 201310008022 A CN201310008022 A CN 201310008022A CN 103222478 A CN103222478 A CN 103222478A
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Abstract
The invention relates to the field of microbial technology and especially relates to a use of Streptomyces sioyaensis metabolite in prevention and control of Fusarium oxyspirum f. sp. niveum. Llex cornuta endophyte separated from the llex cornuta living bodies in Hangzhou of Zhejiang province by an endophyte microorganism separation technology is identified as Streptomyces sioyaensis by microbial taxonomy and is named as Streptomyces sioyaensis 107. The Streptomyces sioyaensis 107 is preserved in the China general microbiological culture collection center on December 12, 2012 and has a preservation number of CGMCC No. 6984. Through liquid fermentation, the Streptomyces sioyaensis 107 can produce the active metabolites having effects of inhibiting plant fungal disease pathogenic bacteria such as Rhizoctonia solani and Thanatephorus cucumeris and is an important microbe resource in agriculture.
Description
Technical field
The present invention relates to microbial technology field, particularly relate to living actinomycetes---the cultivation of salt room streptomycete (Streptomyces sioyaensis) and the application of metabolite thereof in the biological and ecological methods to prevent plant disease, pests, and erosion.
Background technology
All have a large amount of endophytes in the plant corpus, endophyte is found in herbage the earliest, is found in plants such as woody plant, herbaceous plant, gramineae plant, liver moss, lichens, marine alga again afterwards.Studies show that so far, endophyte of plant can strengthen the host disease resistance, improve the productivity, degeneration-resistant pest-resistant etc. of plant.Bringing into play these effects is because endophyte is bred in plant corpus and grown, and generation has bioactive secondary metabolite in vivo.Therefore, endophyte may become potential biocontrol agent in the biological control, has important purposes aspect the ecological agriculture and the biopesticide development.Endophyte of plant has caused microbiologist, ecologist, plant protection scholar's extensive interest in recent years, becomes the focus of domestic and international research gradually.The research and development of microbial pesticide at present is very active in China, but microbial resources and application potential thereof still do not fully played, and endophyte of plant promptly is the untapped emerging microbial resources of such class.
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 of endophyte generation to carry out the biological control of plant pathogenic fungi disease, then have the cycle weak point, be easy to research, be convenient to advantages such as production and low toxicity.
The present invention is separated to living actinomycetes in the strain biological and ecological methods to prevent plant disease, pests, and erosion from Chinese holly, and this strain fermentation metabolite has been carried out bacteriostasis research.This invention provides good starting strain 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, the interior living actinomycetes that provide a strain from Chinese holly, to separate---salt room streptomycete (Streptomyces sioyaensis) at chemical pesticide; Another object of the present invention provides the method for utilizing this bacterial strain metabolite control fungal diseases of plants.
The separation of salt of the present invention room streptomycete, screening and evaluation:
Be in July, 2012 in the Chinese holly stem that Zhejiang Hangzhou is gathered, obtain and preserve through steps such as separation, purifying, cultivation, fermentation and determinations of activity; Be accredited as salt room streptomycete (Streptomyces sioyaensis) through microbial taxonomy, name and be salt room streptomycete 107.This bacterial strain is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation address, preservation date on December 12nd, 2012, preservation registration number CGMCC No.6984.
Salt room streptomycete 107, the microscopic morphology feature: fibrillae of spores is straight, spirality, and spore is oval or oval.
Salt of the present invention room streptomycete 107 can obtain the inhibited metabolite of part fungal diseases of plants pathogen through fermented extracted.
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 metabolic products such as rice sheath blight disease, cucumber rhizoctonia rot, graw mold of tomato, cucumber fusarium axysporum or watermelon blight, carry out according to the following steps:
(1) activation culture of bacterial classification: will separate salt room streptomycete 107 bacterial classifications of preserving and move on potato dextrose agar (PDA) the inclined-plane flat board, and under 28 ℃ ± 1 ℃ condition, be cultured to the test tube slant and cover with spore, and use for inoculation;
(2) fermented and cultured: fermentation culture is to contain soybean cake powder 20g among every 1000mL, corn flour 15g, wheat bran 10g, peptone 5g, (NH
4)
2SO
45g, CaCO
35g, MgSO
410g, the bottled fermentation culture 60mL of every 300mL triangle; Prepare back 121 ℃ of sterilization 20min, to be cooled to 40 ℃ of picking one platinum ring salt room streptomycete 107 spore inoculatings under aseptic condition, under 28 ℃ ± 1 ℃ condition, 180 rev/mins of shaking speed, fermented and cultured 128h;
(3) fermentation product extracts: fermentation finishes, centrifugal collection fermented supernatant fluid, and learning from else's experience with conventional organic solvent extracting obtains medicinal extract behind the vacuum concentration, is used for bacteriostatic activity mensuration.
Beneficial effect of the present invention:
The one, the present invention separates metabolite part fungal diseases of plants pathogen is had stronger inhibiting salt room streptomycete 107 from plant Chinese holly stem, and this provides a strain microbial strains for control fungal diseases of plants pathogen on the agricultural;
The 2nd, the invention provides the method for utilizing 107 fermentations of salt room streptomycete to obtain active metabolite; This metabolite can be applicable to the control of fungal diseases of plants, for the exploitation of disinfectant use in agriculture has increased new approach.
Embodiment
Separation and the screening of embodiment 1:(salt room streptomycete 107)
The present invention gathers the Chinese holly stem in July, 2012 in Zhejiang Hangzhou, through surface sterilization, endophyte separation, cultivation, fermentation, determination of activity and to suppressing the steps such as screening of fungal diseases of plants pathogen active bacterial strain, therefrom obtains salt room streptomycete 107, preserves.
Salt room streptomycete 107 bacteriostatic activities are measured: the fermented supernatant fluid 1mL of the 0.22 μ m membrane filtration of learning from else's experience is in sterile petri dish, be cooled to 50 ℃ the rapid mixing of PDA medium with 9mL, the Rhizoctonia solani Kuhn (Thanatephorus cucumeris) that it is 4mm that the cooling back is put 1 diameter respectively on each medium plane or cucumber fusarium axysporum (Fusarium oxysporum.sp.cucumebrium Owen) or cucumber rhizoctonia rot bacterium (Rhizoctonia solani) or withered germ of water-melon (F.oxysporum f.sp.niveum) or botrytis cinerea (Botrytis cinerea) 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 72h 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: salt room streptomycete 107 metabolites are respectively 91.5%, 88.5%, 82.5%, 86.4 and 86.1% to the inhibiting rate of Rhizoctonia solani Kuhn, cucumber rhizoctonia rot bacterium, botrytis cinerea, cucumber fusarium axysporum and withered germ of water-melon.
The preparation method of embodiment 2:(salt room streptomycete 107 fermentating metabolism products)
Carry out according to the following steps:
(1) activation culture of bacterial classification: will separate salt room streptomycete 107 bacterial classifications of preserving and move on the potato glucose PDA solid plate, and under 28 ℃ ± 1 ℃ condition, be cultured to the test tube slant and cover with spore, and use for inoculation;
(2) fermented and cultured: fermentation culture is to contain soybean cake powder 20g among every 1000mL, corn flour 15g, wheat bran 10g, peptone 5g, (NH
4)
2SO
45g, CaCO
35g, MgSO
410g, the bottled fermentation culture 60mL of every 300mL triangle; Prepare back 121 ℃ of sterilization 20min, to be cooled to 40 ℃ of picking one platinum ring salt room streptomycete 107 spore inoculatings under aseptic condition, under 28 ℃ ± 1 ℃ condition, 180 rev/mins of shaking speed, fermented and cultured 128h;
(3) fermentation product extracts: centrifugal collection zymotic fluid after fermentation finishes, add the equal-volume ethyl acetate extraction, and take off layer, use the equal-volume extracting n-butyl alcohol again, get the upper strata, under vacuum condition, be concentrated into medicinal extract, get n-butyl alcohol extract, measure for bacteriostatic activity.
Salt room streptomycete 107 metabolites described in following examples 3,4 are embodiment 2 products made therebies; The percentage composition of described product is mass/volume percentage.
Embodiment 3:(salt room streptomycete 107 metabolites are to the inhibitory action of Rhizoctonia solani Kuhn, cucumber rhizoctonia rot bacterium, botrytis cinerea, cucumber fusarium axysporum and withered germ of water-melon)
(1) accurately take by weighing salt room streptomycete 107 metabolite 0.20g, and add the sterile water of 10mL volume, ultrasonic oscillation fully dissolves, and finally is configured to the mother liquor that concentration is 20g/L;
(2) bacteriostatic activity is measured: get above-mentioned mother liquor and be diluted to the solution that concentration is 2g/L and 1g/L respectively, get each concentration solution 1mL in sterile petri dish, be cooled to 50 ℃ the rapid mixing of PDA medium (salt room streptomycete 107 metabolite final concentrations are 200mg/L and 100mg/L) with 9mL, the Rhizoctonia solani Kuhn that it is 4mm that the cooling back is put 1 diameter respectively on each medium plane or the bacterium cake of cucumber rhizoctonia rot bacterium or botrytis cinerea or cucumber fusarium axysporum or withered germ of water-melon, 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 conventional chemical medicament and sterile water, repeat 3 times; Adopt the right-angled intersection method to measure colony diameter, calculate inhibiting rate with following formula:
Test example: (product of the present invention and conventional pesticide control efficiency comparative trial)
Result of the test sees Table 1.As seen from Table 1, it is similar or slightly excellent than the conventional chemical medicament that salt room streptomycete 107 metabolites suppress effect, and safety.
Table 1 salt room streptomycete 107 metabolites are to the inhibition effect for the examination disease fungus
"-" expression not test (N.T.).
Claims (1)
1. the application of salt room streptomycete (Streptomyces sioyaensis) 107 metabolites control withered germ of water-melon (Fusarium oxysporumf.sp.niveum), described salt room streptomycete 107 is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preservation date on December 12nd, 2012, preservation registration number CGMCC No.6984, described metabolite prepares by the following method:
(1) activation culture of bacterial classification: will separate salt room streptomycete 107 bacterial classifications of preserving and move on potato dextrose agar (PDA) the inclined-plane flat board, and under 28 ℃ ± 1 ℃ condition, be cultured to the test tube slant and cover with spore, and use for inoculation;
(2) fermented and cultured: fermentation culture is to contain soybean cake powder 20g among every 1000mL, corn flour 15g, wheat bran 10g, peptone 5g, (NH
4)
2SO
45g, CaCO
35g, MgSO
410g, the bottled fermentation culture 60mL of every 300mL triangle; Prepare back 121 ℃ of sterilization 20min, to be cooled to 40 ℃ of picking one platinum ring salt room streptomycete 107 spore inoculatings under aseptic condition, under 28 ℃ ± 1 ℃ condition, 180 rev/mins of shaking speed, fermented and cultured 128h;
(3) fermentation product extracts: centrifugal collection zymotic fluid after fermentation finishes, add the equal-volume ethyl acetate extraction, and take off layer, use the equal-volume extracting n-butyl alcohol again, get the upper strata, under vacuum condition, be concentrated into medicinal extract, get n-butyl alcohol extract.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104342389A (en) * | 2014-09-29 | 2015-02-11 | 中国计量学院 | Streptomyces strains and joint application thereof in prevention and treatment of cucumber fusarium wilt |
Citations (3)
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|---|---|---|---|---|
| US6100053A (en) * | 1994-08-26 | 2000-08-08 | Novo Nordisk A/S | Microbial transglutaminases, their production and use |
| CN102204571A (en) * | 2011-04-08 | 2011-10-05 | 中国计量学院 | Application of Alternaria alternata metabolic product in controlling watermelon fusarium oxysporum |
| CN102210331A (en) * | 2011-04-08 | 2011-10-12 | 中国计量学院 | Application of Nigrospora oryzae 46 metabolic product on preventing and treating Rhizoctonia solani |
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2013
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6100053A (en) * | 1994-08-26 | 2000-08-08 | Novo Nordisk A/S | Microbial transglutaminases, their production and use |
| CN102204571A (en) * | 2011-04-08 | 2011-10-05 | 中国计量学院 | Application of Alternaria alternata metabolic product in controlling watermelon fusarium oxysporum |
| CN102210331A (en) * | 2011-04-08 | 2011-10-12 | 中国计量学院 | Application of Nigrospora oryzae 46 metabolic product on preventing and treating Rhizoctonia solani |
Non-Patent Citations (2)
| Title |
|---|
| PENGJUN SHI 等: "Cloning, characterization, and antifungal activity of an endo-1, 3-β-d-glucanase from Streptomyces sp. S27", 《APPL MICROBIOL BIOTECHNOL》 * |
| TANG-YAO HONG 等: "Isolation and biochemical characterization of an endo-1,3-β-glucanase from Streptomyces sioyaensis containing a C-terminal family 6 carbohydrate-binding module that binds to 1,3-β-glucan", 《MICROBIOLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104342389A (en) * | 2014-09-29 | 2015-02-11 | 中国计量学院 | Streptomyces strains and joint application thereof in prevention and treatment of cucumber fusarium wilt |
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