CN103222482B - Use of Streptomyces sioyaensis metabolite in prevention and control of Alternaria solani - Google Patents
Use of Streptomyces sioyaensis metabolite in prevention and control of Alternaria solani Download PDFInfo
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- CN103222482B CN103222482B CN201310008059.0A CN201310008059A CN103222482B CN 103222482 B CN103222482 B CN 103222482B CN 201310008059 A CN201310008059 A CN 201310008059A CN 103222482 B CN103222482 B CN 103222482B
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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 Alternaria solani. 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 active metabolites having effects of inhibiting plant fungal disease pathogenic bacteria such as Colletotrichum lindemuthianum and Alternaria solani and is an important microbe resource in agriculture.
Description
Technical field
The present invention relates to microbial technology field, particularly relate to biological and ecological methods to prevent plant disease, pests, and erosion endogeny rayungus---the cultivation of salt room streptomycete (Streptomyces sioyaensis) and the application of metabolite thereof.
Background technology
All there is a large amount of endophytes in plant corpus, endophyte is found in herbage the earliest, is found again afterwards in the plants such as woody plant, herbaceous plant, gramineae plant, liver moss, lichens, marine alga.Research so far shows, endophyte of plant can strengthen the disease resistance of host, improve the productivity, degeneration-resistant pest-resistant etc. of plant.Playing these effects is due to endophyte Growth and Reproduction in plant corpus, and generation has bioactive secondary metabolite in vivo.Therefore, endophyte may become potential biocontrol agent in biological control, in the ecological agriculture and biopesticide development, have important purposes.Endophyte of plant has caused the broad interest of microbiologist, ecologist, plant protection scholar in recent years, becomes the focus of research both at home and abroad gradually.The research and development of current microbial pesticide is very active in China, but microbial resources and application potential thereof are not still fully played, and namely endophyte of plant is the untapped emerging microbial resources of such class.
Control at present to plant disease, pathogen killed by the main chemical pesticide that adopts, but chemical pesticide is still difficult to effective solution so far to the toxic and side effect of people and animals and residue problem.The active substance of the disease-resistant fungal pathogens utilizing endophyte to produce carries out the biological control of plant pathogenic fungi disease, then have the cycle short, be easy to research, be convenient to produce and the advantage such as low toxicity.
Summary of the invention
The present invention is separated to a strain biological and ecological methods to prevent plant disease, pests, and erosion endogeny rayungus from Chinese holly, and has carried out bacteriostasis research to this strain fermentation metabolite.The further Study and Development that this invention is microbial pesticide provides excellent starting strain.
The object of the invention is, for the dangerous deficiency less with microbial pesticide kind of chemical pesticide, to provide the endogeny rayungus that a strain separates from Chinese holly---salt room streptomycete (Streptomyces sioyaensis); Another object of the present invention is to provide the method utilizing this Metabolite to prevent and treat fungal diseases of plants.
The separation of salt room of the present invention streptomycete, selection systems:
Be in the Chinese holly stem gathered in Zhejiang Hangzhou in July, 2012, obtain through steps such as separation, purifying, cultivation, fermentation and determinations of activity and preserve; Be accredited as salt room streptomycete (Streptomyces sioyaensis) through microbial taxonomy, name as salt room streptomycete 107.This bacterial strain in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, 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, micro-morphology: fibrillae of spores is straight, spirality, and spore is oval or oval.
Salt room of the present invention streptomycete 107, extracts through fermentation and can obtain the inhibited metabolite of Activities of Some Plants fungal disease pathogen.
The object of the invention is achieved through the following technical solutions:
Prevent and treat a preparation method for the fungal diseases of plants such as bean anthracnose or early blight of tomato microbial metabolic products, carry out according to the following steps:
(1) activation culture of bacterial classification: moving to being separated salt room streptomycete 107 bacterial classification preserved on potato dextrose agar (PDA) inclined-plane flat board, being cultured to test tube slant and covering with spore under 28 DEG C ± 1 DEG C condition, for inoculation;
(2) fermented and cultured: fermentation culture is containing soybean cake powder 20g, corn flour 15g, wheat bran 10g in every 1000mL, peptone 5g, (NH
4)
2sO
45g, CaCO
35g, MgSO
410g, the bottled fermentation culture 60mL of every 300mL triangle; Prepare rear 121 DEG C of sterilizing 20min, to be cooled to 40 DEG C of aseptically picking one platinum ring salt room streptomycete 107 spore inoculatings, under 28 DEG C ± 1 DEG C condition, shaking speed 180 revs/min, fermented and cultured 128h;
(3) fermentation product extracts: ferment complete, collected by centrifugation fermented supernatant fluid, obtains medicinal extract, for Antibacterial Activity with conventional organic solvent extraction after Vacuum Concentration.
Beneficial effect of the present invention:
One is that the present invention separates metabolite and has salt room streptomycete 107 compared with high inhibition effect to Activities of Some Plants fungal disease pathogen from plant Chinese holly stem, and this provides a strain microbial strains for agriculturally preventing and treating fungal diseases of plants pathogen;
Two is the invention provides the method utilizing streptomycete 107 fermentation of salt room to obtain active metabolite; This metabolite can be applicable to the control of fungal diseases of plants, and the exploitation for disinfectant use in agriculture adds new approach.
Embodiment
The isolation and screening of embodiment 1:(salt room streptomycete 107)
The present invention gathers Chinese holly stem in July, 2012 in Zhejiang Hangzhou, be separated through surface sterilization, endophyte, cultivate, fermentation, determination of activity and to the step such as screening suppressing fungal diseases of plants pathogen active bacterial strain, therefrom obtain salt room streptomycete 107, preserve.
Salt room streptomycete 107 Antibacterial Activity: the fermented supernatant fluid 1mL of 0.22 μm of membrane filtration of learning from else's experience is in sterile petri dish, the PDA medium being cooled to 50 DEG C with 9mL mixes rapidly, bean anthrax bacteria (Colletotrichum lindemuthianum) that 1 diameter is 4mm or tomato early blight bacterium (Alternaria solani) etc. are put respectively for examination bacterium bacterium cake in each medium plane after cooling, bacterium cake is connected to culture dish central authorities (culture dish diameter is 9cm), put 28 DEG C of cultivation 72h in incubator, with sterile water process in contrast, repeat 3 times; Right-angled intersection method is adopted to measure colony diameter, with following formulae discovery inhibiting rate:
Measurement result shows: the salt room inhibiting rate of streptomycete 107 metabolite to bean anthrax bacteria and tomato early blight bacterium is respectively 89.1% and 86.8%.
The preparation method of embodiment 2:(salt room streptomycete 107 fermentating metabolism product)
Carry out according to the following steps:
(1) activation culture of bacterial classification: moving on potato glucose PDA solid plate by being separated salt room streptomycete 107 bacterial classification preserved, being cultured to test tube slant and covering with spore under 28 DEG C ± 1 DEG C condition, for inoculation;
(2) fermented and cultured: fermentation culture is containing soybean cake powder 20g, corn flour 15g, wheat bran 10g in every 1000mL, peptone 5g, (NH
4)
2sO
45g, CaCO
35g, MgSO
410g, the bottled fermentation culture 60mL of every 300mL triangle; Prepare rear 121 DEG C of sterilizing 20min, to be cooled to 40 DEG C of aseptically picking one platinum ring salt room streptomycete 107 spore inoculatings, under 28 DEG C ± 1 DEG C condition, shaking speed 180 revs/min, fermented and cultured 128h;
(3) fermentation product extracts: collected by centrifugation zymotic fluid after fermentation, adds equal-volume extraction into ethyl acetate, take off layer, then uses equal-volume extracting n-butyl alcohol, gets upper strata, is concentrated into medicinal extract under vacuum, obtain n-butyl alcohol extract, for Antibacterial Activity.
Salt room streptomycete 107 metabolite described in following examples 3,4 is embodiment 2 products made thereby; The percentage composition of described product is mass/volume percentage.
Embodiment 3:(salt room streptomycete 107 metabolite is to the inhibitory action of bean anthrax bacteria or tomato early blight bacterium)
(1) accurately take salt room streptomycete 107 metabolite 0.20g, and add the sterile water of 10mL volume, ultrasonic oscillation fully dissolves, and is finally configured to the mother liquor that concentration is 20g/L;
(2) Antibacterial Activity: get above-mentioned mother liquor and be diluted to the solution that concentration is 2g/L and 1g/L respectively, get each strength solution 1mL in sterile petri dish, the PDA medium being cooled to 50 DEG C with 9mL mixes rapidly (salt room streptomycete 107 metabolite final concentration is 200mg/L and 100mg/L), putting 1 diameter respectively in each medium plane after cooling is the bean anthrax bacteria of 4mm or the bacterium cake of tomato early blight bacterium, bacterium cake is connected to culture dish central authorities (culture dish diameter is 9cm), put 28 DEG C of cultivation 36h in incubator, with conventional chemical medicament and sterile water process in contrast, repeat 3 times, right-angled intersection method is adopted to measure colony diameter, with following formulae discovery inhibiting rate:
Test example: (product of the present invention and conventional pesticide control efficiency comparative trial)
Result of the test is in table 1.As seen from Table 1, salt room streptomycete 107 metabolite inhibition is similar compared with conventional chemical medicament or slightly excellent, and safety.
Table 1 salt room streptomycete 107 metabolite is to the inhibition for examination disease fungus
"-" represents not test (N.T.).
Claims (1)
1. the application of salt room streptomycete (Streptomyces sioyaensis) 107 metabolite control tomato early blight bacterium (Alternaria solani), described salt room streptomycete 107 is in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preservation date on December 12nd, 2012, preservation registration number CGMCC No.6984, described metabolite is prepared by the following method:
(1) activation culture of bacterial classification: moving to being separated salt room streptomycete 107 bacterial classification preserved on potato dextrose agar (PDA) inclined-plane flat board, being cultured to test tube slant and covering with spore under 28 DEG C ± 1 DEG C condition, for inoculation;
(2) fermented and cultured: fermentation culture is containing soybean cake powder 20g, corn flour 15g, wheat bran 10g in every 1000mL, peptone 5g, (NH
4)
2sO
45g, CaCO
35g, MgSO
410g, the bottled fermentation culture 60mL of every 300mL triangle; Prepare rear 121 DEG C of sterilizing 20min, to be cooled to 40 DEG C of aseptically picking one platinum ring salt room streptomycete 107 spore inoculatings, under 28 DEG C ± 1 DEG C condition, shaking speed 180 revs/min, fermented and cultured 128h;
(3) fermentation product extracts: collected by centrifugation zymotic fluid after fermentation, adds equal-volume extraction into ethyl acetate, take off layer, then uses equal-volume extracting n-butyl alcohol, gets upper strata, is concentrated into medicinal extract under vacuum, obtain n-butyl alcohol extract.
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| CN102204570A (en) * | 2011-04-08 | 2011-10-05 | 中国计量学院 | Application of alternaria alternate metabolic products in cucumber rhizoctonia solani prevention and control |
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| DE1250598B (en) * | 1967-09-21 | Eli Lilly and Company Indmapolis, Ind (V St A) | I Process for fermentative Her position of Al 16SO and Al 16SA designated antibiotic, in particular fungicidally acting peptides | |
| JPS5868758A (en) * | 1981-10-20 | 1983-04-23 | Konishiroku Photo Ind Co Ltd | Developing method for electrostatic image |
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| CN102204570A (en) * | 2011-04-08 | 2011-10-05 | 中国计量学院 | Application of alternaria alternate metabolic products in cucumber rhizoctonia solani prevention and control |
Non-Patent Citations (2)
| Title |
|---|
| JP平1-26057B2 1989.05.22 * |
| 鏈黴菌 Streptomyces sioyaensis PMS502 量產及其於病害防治之應用;何炳璋;《国立中兴大学硕士学位论文》;20011231;摘要 * |
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