CN103222479A - Use of Streptomyces sioyaensis metabolite in prevention and control of Colletotrichum lindemuthianum - Google Patents
Use of Streptomyces sioyaensis metabolite in prevention and control of Colletotrichum lindemuthianum Download PDFInfo
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- CN103222479A CN103222479A CN2013100080285A CN201310008028A CN103222479A CN 103222479 A CN103222479 A CN 103222479A CN 2013100080285 A CN2013100080285 A CN 2013100080285A CN 201310008028 A CN201310008028 A CN 201310008028A CN 103222479 A CN103222479 A CN 103222479A
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- 239000002207 metabolite Substances 0.000 title claims abstract description 19
- 241000946750 Streptomyces sioyaensis Species 0.000 title claims abstract description 8
- 230000002265 prevention Effects 0.000 title claims 2
- 241001120669 Colletotrichum lindemuthianum Species 0.000 title abstract description 3
- 238000000855 fermentation Methods 0.000 claims abstract description 18
- 230000004151 fermentation Effects 0.000 claims abstract description 18
- 238000004321 preservation Methods 0.000 claims abstract description 7
- 241000187747 Streptomyces Species 0.000 claims abstract 3
- 239000007788 liquid Substances 0.000 claims abstract 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 10
- 239000000284 extract Substances 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
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- 235000010469 Glycine max Nutrition 0.000 claims description 3
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- 240000008042 Zea mays Species 0.000 claims description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 3
- 235000005822 corn Nutrition 0.000 claims description 3
- 235000013312 flour Nutrition 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims 3
- 241000222199 Colletotrichum Species 0.000 claims 1
- 239000012675 alcoholic extract Substances 0.000 claims 1
- 239000012141 concentrate Substances 0.000 claims 1
- 238000000605 extraction Methods 0.000 claims 1
- 239000001963 growth medium Substances 0.000 claims 1
- 238000009629 microbiological culture Methods 0.000 claims 1
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- 244000005700 microbiome Species 0.000 abstract description 4
- 241000213004 Alternaria solani Species 0.000 abstract description 2
- 241000207199 Citrus Species 0.000 abstract 1
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- 235000020971 citrus fruits Nutrition 0.000 abstract 1
- 241001576300 endophytic bacterium Species 0.000 abstract 1
- 241001655322 Streptomycetales Species 0.000 description 25
- 150000003839 salts Chemical class 0.000 description 22
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- 241000894006 Bacteria Species 0.000 description 8
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- 244000046052 Phaseolus vulgaris Species 0.000 description 6
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 6
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- 241001465754 Metazoa Species 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
本发明涉及微生物技术领域，本发明所述的枸骨内生菌是从浙江杭州采集的枸骨植物活体中采用内生菌微生物分离技术分离获得的，经微生物分类学鉴定为盐屋链霉菌(Streptomyces sioyaensis)，定名为盐屋链霉菌107。该菌株已在中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏日期2012年12月12日，保藏登记号CGMCC No.6984。本发明最显著的特征是该菌株液体发酵能够产生对植物真菌病害病原菌如菜豆炭疽病菌(Colletotrichum lindemuthianum)和番茄早疫病菌(Alternaria solani)等有抑制作用的活性代谢产物，是农业上重要的微生物资源。The present invention relates to the technical field of microbes. The endophytic bacterium of the Chinese chinensis described in the present invention is obtained from the living body of the Chinese citrus bone collected in Hangzhou, Zhejiang by adopting an endophytic microorganism separation technology, and is identified as Streptomyces shiiyaki ( Streptomyces sioyaensis), named Streptomyces sioyaensis 107. The strain has been preserved in the General Microorganism Center of the China Committee for the Collection of Microorganisms, with a preservation date of December 12, 2012, and a preservation registration number of CGMCC No.6984. The most notable feature of the present invention is that the liquid fermentation of the strain can produce active metabolites that can inhibit plant fungal disease pathogens such as Colletotrichum lindemuthianum and Alternaria solani, and are important microorganisms in agriculture. resource.
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 bean anthracnose or early blight of tomato, 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, bean anthrax bacteria (Colletotrichum lindemuthianum) that it is 4mm that the cooling back is put 1 diameter respectively on each medium plane or tomato early blight bacterium (Alternaria solani) 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 89.1% and 86.8% to the inhibiting rate of bean anthrax bacteria and tomato early blight bacterium.
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 bean anthrax bacteria or tomato early blight bacterium)
(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 bean anthrax bacteria that it is 4mm that the cooling back is put 1 diameter respectively on each medium plane or the bacterium cake of tomato early blight bacterium, 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)
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| CN201310008028.5A CN103222479B (en) | 2013-01-07 | 2013-01-07 | Use of Streptomyces sioyaensis metabolite in prevention and control of Colletotrichum lindemuthianum |
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|---|---|---|---|
| CN201310008028.5A CN103222479B (en) | 2013-01-07 | 2013-01-07 | Use of Streptomyces sioyaensis metabolite in prevention and control of Colletotrichum lindemuthianum |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103952362A (en) * | 2014-05-14 | 2014-07-30 | 浙江省柑桔研究所 | Citrus endophytic actinomycetes with antibacterial activity on various plant pathogens |
| 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)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60156392A (en) * | 1984-01-25 | 1985-08-16 | Fujisawa Pharmaceut Co Ltd | Fr-900336 substance and its preparation |
| US6100053A (en) * | 1994-08-26 | 2000-08-08 | Novo Nordisk A/S | Microbial transglutaminases, their production and use |
| CN102204572A (en) * | 2011-04-08 | 2011-10-05 | 中国计量学院 | Use of metabolites of nigrospora oryzae 46 in prevention of colletotrichum lindemuthianum |
-
2013
- 2013-01-07 CN CN201310008028.5A patent/CN103222479B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60156392A (en) * | 1984-01-25 | 1985-08-16 | Fujisawa Pharmaceut Co Ltd | Fr-900336 substance and its preparation |
| US6100053A (en) * | 1994-08-26 | 2000-08-08 | Novo Nordisk A/S | Microbial transglutaminases, their production and use |
| CN102204572A (en) * | 2011-04-08 | 2011-10-05 | 中国计量学院 | Use of metabolites of nigrospora oryzae 46 in prevention of colletotrichum lindemuthianum |
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》, vol. 85, 31 December 2010 (2010-12-31), pages 1483 - 1490, XP019778534 * |
| 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》, vol. 148, no. 4, 30 April 2002 (2002-04-30), pages 1151 - 1159 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103952362A (en) * | 2014-05-14 | 2014-07-30 | 浙江省柑桔研究所 | Citrus endophytic actinomycetes with antibacterial activity on various plant pathogens |
| CN103952362B (en) * | 2014-05-14 | 2015-12-30 | 浙江省柑桔研究所 | One strain is to the oranges and tangerines endogeny rayungus of various plants pathogenic bacteria tool bacteriostatic activity |
| 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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