CN103087949B - Biocontrol endophytic actinomycetes- streptomyces sioyaensis - Google Patents
Biocontrol endophytic actinomycetes- streptomyces sioyaensis Download PDFInfo
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- CN103087949B CN103087949B CN201310008054.8A CN201310008054A CN103087949B CN 103087949 B CN103087949 B CN 103087949B CN 201310008054 A CN201310008054 A CN 201310008054A CN 103087949 B CN103087949 B CN 103087949B
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- endophyte
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- room streptomycete
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Abstract
The invention relates to the technical field of microorganisms. Chinese holly endophyte disclosed by the invention is obtained by adopting an endophyte microorganism separation technology for separating a Chinese holly plant living body collected in Hangzhou of Zhejiang Province, is identified as streptomyces sioyaensis according to microbial taxonomy and is named as streptomyces sioyaensis107. The strain is preserved in the China General Microbiological Culture Collection Center with the preservation date of December 12th of 2012 and the preservation register number of CGMCC No.6984. The prominent characteristic of the strain is as follows: through fermentation, the liquid of the strain can generate active metabolite which has an inhibition effect on fungal diseases and pathogenic bacteria of plants such as Rhizoctonia solani of cucumber and hanatephorus cucumeris; and the strain is an important agricultural microorganism resource.
Description
Technical field
The present invention relates to microbial technology field, particularly relate to raw actinomycetes---the cultivation of salt room streptomycete (Streptomyces sioyaensis) and the application of meta-bolites thereof in biological and ecological methods to prevent plant disease, pests, and erosion.
Background technology
In plant materials, all have a large amount of endophytes, endophyte is found in herbage the earliest, is found again afterwards in the plants such as xylophyta, herbaceous plant, gramineae plant, liver moss, lichens, marine alga.Research so far shows, endophyte of plant can strengthen host disease resistance, improve the productivity, degeneration-resistant pest-resistant etc. of plant.Bringing into play these effects is due to endophyte Growth and Reproduction in plant materials, and produces and have bioactive secondary metabolite in vivo.Therefore, endophyte may become potential biocontrol agent in biological control, aspect the ecological agriculture and biological pesticide development, has important purposes.Endophyte of plant has caused microbiologist, ecologist, plant protection scholar's broad interest in recent years, becomes gradually the focus of domestic and international research.The at present research and development of microbial pesticide is very active in China, but Microbial resources and application potential thereof still do not fully played, and endophyte of plant is the untapped emerging Microbial resources of such class.
Control to Plant diseases, mainly adopt chemical pesticide to kill pathogenic bacteria, but toxic side effect and the residue problem of chemical pesticide to people and animals is still difficult to effective solution so far at present.The active substance of disease-resistant fungal pathogens that utilizes endophyte to produce carries out the biological control of plant pathogenic fungi disease, have the cycle short, be easy to research, be convenient to produce and the advantage such as low toxicity.
The present invention is separated to raw actinomycetes in a strain biological and ecological methods to prevent plant disease, pests, and erosion from Chinese holly, and this strain fermentation meta-bolites has been carried out to bacteriostatic action research.Further development and exploitation that this invention is microbial pesticide provide good starting strain.
Summary of the invention
The interior raw actinomycetes that provide a strain to separate from Chinese holly---salt room streptomycete (Streptomyces sioyaensis) is provided for the deficiency that chemical pesticide is dangerous and microbial pesticide kind is less; Another object of the present invention is to provide the method for utilizing this Metabolite control fungal diseases of plants.
Separation, screening and the qualification of salt of the present invention room streptomycete:
Be in the Chinese holly stem gathering in Zhejiang Hangzhou in July, 2012, 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 as salt room streptomycete 107.This bacterial strain 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.
Salt room streptomycete 107, micro-morphology: fibrillae of spores is straight, volution, and spore is oval or oval.
Salt of the present invention room streptomycete 107, can obtain the inhibited meta-bolites of Activities of Some Plants fungal disease pathogenic bacteria through fermented extracted.
The object of the invention is achieved through the following technical solutions:
A preparation method who prevents and treats the fungal diseases of plants microbial metabolites such as rice sheath blight disease, cucumber rhizoctonia rot, graw mold of tomato, cucumber fusarium axysporum or watermelon blight, carries out according to the following steps:
(1) activation culture of bacterial classification: move on potato dextrose agar (PDA) inclined-plane flat board separating salt room streptomycete 107 bacterial classifications of preserving, be cultured to test tube slant and cover with spore under 28 DEG C ± 1 DEG C condition, for inoculation;
(2) fermentation culture: fermentation culture is to contain soybean cake powder 20g in every 1000mL, Semen Maydis powder 15g, wheat bran 10g, 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 picking one platinum ring salt room streptomycete 107 spore inoculatings under aseptic condition, under 28 DEG C ± 1 DEG C condition, 180 revs/min of shaking speed, fermentation culture 128h;
(3) fermented product extracts: ferment complete, centrifugal collection fermented supernatant fluid, learns from else's experience after vacuum concentration and obtain medicinal extract with conventional organic solvent extracting, for Antibacterial Activity.
Beneficial effect of the present invention:
The one, the present invention separates metabolite Activities of Some Plants fungal disease pathogenic bacteria is had to stronger inhibiting salt room streptomycete 107 from plant Chinese holly stem, and this provides a strain microorganism strains for preventing and treating fungal diseases of plants pathogenic bacteria in agricultural;
The 2nd, the invention provides the method for utilizing 107 fermentations of salt room streptomycete to obtain active metabolite; This meta-bolites can be applicable to the control of fungal diseases of plants, for the exploitation of disinfectant use in agriculture has increased 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, through steps such as surface sterilization, endophyte separation, cultivation, fermentation, determination of activity and the screenings to inhibition fungal diseases of plants pathogenic bacteria active bacterial strain, therefrom obtains salt room streptomycete 107, preserves.
Salt room streptomycete 107 Antibacterial Activities: the fermented supernatant fluid 1mL of the 0.22 μ m membrane filtration of learning from else's experience is in sterile petri dish, the PDA substratum that is cooled to 50 DEG C with 9mL mixes rapidly, after cooling, put respectively the Rhizoctonia solani Kuhn that 1 diameter is 4mm (Thanatephorus cucumeris) 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. for examination bacterium bacterium cake in each substratum plane, bacterium cake is connected to culture dish central authorities (culture dish diameter is 9cm), put 28 DEG C of cultivation 72h in incubator, process in contrast with sterilized water, repeat 3 times, adopt right-angled intersection method to measure colony diameter, calculate inhibiting rate with following formula:
Measurement result shows: salt room streptomycete 107 meta-bolitess 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: move on potato glucose PDA solid plate separating salt room streptomycete 107 bacterial classifications of preserving, be cultured to test tube slant and cover with spore under 28 DEG C ± 1 DEG C condition, for inoculation;
(2) fermentation culture: fermentation culture is to contain soybean cake powder 20g in every 1000mL, Semen Maydis powder 15g, wheat bran 10g, 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 picking one platinum ring salt room streptomycete 107 spore inoculatings under aseptic condition, under 28 DEG C ± 1 DEG C condition, 180 revs/min of shaking speed, fermentation culture 128h;
(3) fermented product extracts: centrifugal collection fermented liquid after fermentation, add the extraction of equal-volume ethyl acetate, and take off layer, then use equal-volume n-butanol extraction, get upper strata, under vacuum condition, be concentrated into medicinal extract, obtain n-butyl alcohol extract, for Antibacterial Activity.
Salt room streptomycete 107 meta-bolitess described in following examples 3,4 are embodiment 2 products made therebies; The percentage composition of described product is mass/volume per-cent.
The restraining effect of embodiment 3:(salt room streptomycete 107 meta-bolitess to Rhizoctonia solani Kuhn, cucumber rhizoctonia rot bacterium, botrytis cinerea, cucumber fusarium axysporum and withered germ of water-melon)
(1) accurately take salt room streptomycete 107 meta-bolites 0.20g, and add the sterilized 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 respectively the solution that concentration is 2g/L and 1g/L, get each concentration solution 1mL in sterile petri dish, the PDA substratum that is cooled to 50 DEG C with 9mL mixes rapidly (salt room streptomycete 107 meta-bolites final concentrations are 200mg/L and 100mg/L), after cooling, put respectively Rhizoctonia solani Kuhn that 1 diameter is 4mm or the bacterium cake of cucumber rhizoctonia rot bacterium or botrytis cinerea or cucumber fusarium axysporum or withered germ of water-melon in each substratum plane, bacterium cake is connected to culture dish central authorities (culture dish diameter is 9cm), put 28 DEG C of cultivation 36h in incubator, process in contrast with conventional chemical medicament and sterilized water, repeat 3 times, adopt right-angled intersection method to measure colony diameter, calculate inhibiting rate with following formula:
Test example: (product of the present invention and conventional pesticide prevention effect simultaneous test)
Test-results is in table 1.As seen from Table 1, salt room streptomycete 107 meta-bolites inhibitions are similar or slightly excellent compared with conventional chemical medicament, and safety.
Table 1 salt room streptomycete 107 meta-bolitess are to the inhibition for examination pathogenic fungi
"-" represents not test (N.T.).
Claims (1)
1. a strain salt room streptomycete bacterial strain, it is characterized in that, called after salt room streptomycete (Streptomyces sioyaensis) 107, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation day: on December 12nd, 2012, preservation registration number is CGMCC No.6984.
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| CN201310008054.8A CN103087949B (en) | 2013-01-07 | 2013-01-07 | Biocontrol endophytic actinomycetes- streptomyces sioyaensis |
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| CN103087949B true CN103087949B (en) | 2014-07-30 |
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| CN108935520B (en) * | 2015-12-01 | 2019-08-09 | 吉林省林业科学研究院 | A kind of biological prevention and control agent, preparation method and its application method |
| CN116536224B (en) * | 2023-07-03 | 2023-10-03 | 中国农业大学 | Streptomyces salt house Y88 and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101225368A (en) * | 2007-11-19 | 2008-07-23 | 中国计量学院 | Biocontrol actinomyces-streptomyces diastatochromogenes D |
| CN101525587A (en) * | 2009-03-25 | 2009-09-09 | 中国农业大学 | Streptomyces strain and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101225368A (en) * | 2007-11-19 | 2008-07-23 | 中国计量学院 | Biocontrol actinomyces-streptomyces diastatochromogenes D |
| CN101525587A (en) * | 2009-03-25 | 2009-09-09 | 中国农业大学 | Streptomyces strain and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| "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";Tang-Yao Hong;《Microbiology》;20020430;第148卷(第4期);1152页右栏2段 * |
| Tang-Yao Hong."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》.2002,第148卷(第4期), |
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