CN102204572A - Use of metabolites of nigrospora oryzae 46 in prevention of colletotrichum lindemuthianum - Google Patents
Use of metabolites of nigrospora oryzae 46 in prevention of colletotrichum lindemuthianum Download PDFInfo
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- CN102204572A CN102204572A CN2011100882060A CN201110088206A CN102204572A CN 102204572 A CN102204572 A CN 102204572A CN 2011100882060 A CN2011100882060 A CN 2011100882060A CN 201110088206 A CN201110088206 A CN 201110088206A CN 102204572 A CN102204572 A CN 102204572A
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- nigrospora oryzae
- tuber melanosporum
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Abstract
The invention relates to the technical field of microbe. Dogbane endophytic fungus provided by the present invention is isolated from a living dogbane collected in Zhejiang Academy of Agricultural Sciences through an endophytic fungi separation technology. The dogbane endophytic fungus is identified through microbial taxonomy, and is named as nigrospora oryzae 46. The nigrospora oryzae 46 is preserved in the China General Microbiological Culture Collection Center (CGMCC). The preservation date is 2010/12/31, and the preservation register number is CGMCC No.4509. The nigrospora oryzae 46 is characterized in that: the nigrospora oryzae 46 can generate active metabolites through a liquid fermentation, and the active metabolites provide inhibitions for rhizoctonia solani, colletotrichum lindemuthianum and botrytis cinerea. Therefore, the nigrospora oryzae 46 is an important microbe resource in agriculture.
Description
Technical field
The present invention relates to microbial technology field, particularly relate to the biological and ecological methods to prevent plant disease, pests, and erosion endogenetic fungus---the cultivation of rice Tuber Melanosporum (Nigrospora oryzae) and the application of metabolite thereof.
Background technology
All have a large amount of endogenetic fungus in the plant corpus, endogenetic fungus 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, plant endogenesis epiphyte can strengthen the host disease resistance, improve the productivity, degeneration-resistant pest-resistant etc. of plant.Bringing into play these effects is because endogenetic fungus is bred in plant corpus and grown, and generation has bioactive secondary metabolite in vivo.Therefore, endogenetic fungus may become potential biocontrol agent in the biological control, has important purposes aspect the ecological agriculture and the biopesticide development.Plant endogenesis epiphyte 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 source biochemistry agricultural chemicals at present is very active in China, but microbial resources and application potential thereof still do not fully played, and plant endogenesis epiphyte 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 endogenetic fungus 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 a strain biological and ecological methods to prevent plant disease, pests, and erosion endogenetic fungus from sweet-scented oleander, 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, a kind of endogenetic fungus that separates from sweet-scented oleander-rice Tuber Melanosporum (Nigrospora oryzae) is provided 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 rice Tuber Melanosporum of the present invention, screening and evaluation:
Be in the sweet-scented oleander stem of in the Zhejiang Academy of Agricultural Science, gathering in June, 2008, obtain and preservation through steps such as separation, purifying, cultivation, fermentation and determinations of activity; Be accredited as rice Tuber Melanosporum (Nigrospora oryzae) through microbial taxonomy, name and be rice Tuber Melanosporum (Nigrospora oryzae) 46.This bacterial strain is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preservation date on December 31st, 2010, preservation registration number CGMCCNo.4509.
Rice Tuber Melanosporum 46, its solid culture is characterized as: bacterium colony just is white on the PDA medium, and the back is greyish white, the aerial hyphae prosperity.28 ℃ of 4-5 days expansion 9cm on flat board.The microscopic morphology feature: conidiophore is colourless, have every; Conidium black, ovum circle are to capable, the smooth surface, unicellular of ball.
Rice Tuber Melanosporum 46 of the present invention 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 cucumber rhizoctonia rot, graw mold of tomato or bean anthracnose, carry out according to the following steps:
(1) activation culture of bacterial classification: will separate rice Tuber Melanosporum 46 bacterial classifications of preserving and move on the potato glucose PDA solid plate, and under 28 ℃ ± 1 ℃ condition, cultivate, and cover whole culture dish to mycelia, and obtain the bacterium cake with diameter for the punching of 4mm card punch, for inoculation usefulness;
(2) fermented and cultured: fermentation culture is to contain soybean cake powder 20g among every 1000mL, corn flour 15g, soluble starch 20g, wheat bran 10g, peptone 8g, NH
4NO
33g, (NH
4)
2SO
45g, CaCO
35g, MgSO
41g, the bottled fermentation culture 60mL of every 300mL triangle; Prepare back 121 ℃ the sterilization 20min, to be cooled to 40 ℃ under aseptic condition picking 1 diameter be 4mm bacterium cake, 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 rice Tuber Melanosporum 46 from plant sweet-scented oleander stem, and this provides a kind of microbial strains for control fungal diseases of plants pathogen on the agricultural;
The 2nd, the invention provides the method for utilizing 46 fermentations of rice Tuber Melanosporum 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
The separation and the screening of embodiment 1:(rice Tuber Melanosporum 46)
The present invention gathers the sweet-scented oleander stem in the Zhejiang Academy of Agricultural Science in June, 2008, through surface sterilization, endogenetic fungus separation, cultivation, fermentation, determination of activity and to suppressing the steps such as screening of fungal diseases of plants pathogen effective strain, therefrom obtain rice Tuber Melanosporum 46, preserve.
Rice Tuber Melanosporum 46 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 cucumber rhizoctonia rot bacterium (Rhizoctonia solani) that it is 4mm that the cooling back is put 1 diameter respectively on each medium plane or bean anthrax bacteria (Colletotrichum lindemuthianum) 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: rice Tuber Melanosporum 46 metabolites are respectively 96%, 93% and 98% to the inhibiting rate of cucumber rhizoctonia rot bacterium, withered germ of water-melon and botrytis cinerea.
The preparation method of embodiment 2:(rice Tuber Melanosporum 46 fermentating metabolism products)
Carry out according to the following steps:
(1) activation culture of bacterial classification: will separate rice Tuber Melanosporum 46 bacterial classifications of preserving and move on the potato glucose PDA solid plate, and under 28 ℃ ± 1 ℃ condition, cultivate, and cover whole culture dish to mycelia, and obtain the bacterium cake with diameter for the punching of 4mm card punch, for inoculation usefulness;
(2) fermented and cultured: fermentation culture is to contain soybean cake powder 20g among every 1000mL, corn flour 15g, soluble starch 20g, wheat bran 10g, peptone 8g, NH
4NO
33g, (NH
4)
2SO
45g, CaCO
35g, MgSO
41g, the bottled fermentation culture 60mL of every 300mL triangle; Prepare back 121 ℃ the sterilization 20min, to be cooled to 40 ℃ under aseptic condition picking 1 diameter be 4mm bacterium cake, 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.
Rice Tuber Melanosporum 46 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:(rice Tuber Melanosporum 46 metabolites are to the inhibitory action of cucumber rhizoctonia rot bacterium, bean anthrax bacteria and botrytis cinerea)
(1) accurately take by weighing rice Tuber Melanosporum 46 metabolite 0.03g, and add the sterile water of 10mL volume, ultrasonic oscillation fully dissolves, and finally is configured to the mother liquor that concentration is 3g/L;
(2) bacteriostatic activity is measured: get above-mentioned mother liquor and be diluted to the solution that concentration is 1500mg/L and 1000mg/L respectively, get each concentration solution 1mL in sterile petri dish, be cooled to 50 ℃ the rapid mixing of PDA medium (rice Tuber Melanosporum 46 metabolite final concentrations are 150mg/L and 100mg/L) with 9mL, bean anthrax bacteria or cucumber rhizoctonia rot bacterium or botrytis cinerea bacterium cake that it is 4mm that the cooling back is put 1 diameter respectively on each medium plane, 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 slightly excellent than the conventional chemical medicament that rice Tuber Melanosporum 46 metabolites suppress effect, and safety.
Table 1 rice Tuber Melanosporum 46 metabolites are to the inhibition effect for the examination disease fungus
"-" expression not test (N.T.).
Claims (2)
1. the application of rice Tuber Melanosporum (Nigrospora oryzae) 46 metabolites control bean anthrax bacterias (Colletotrichum lindemuthianum).
2. the described application of claim 1, its semilate rice Tuber Melanosporum 46 metabolites are obtained by following method: rice Tuber Melanosporum 46 obtains zymotic fluid through slant activation cultivation, fermented and cultured, add the equal-volume ethyl acetate extraction, 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, described alternaric bacteria 31 is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, and preservation registration number is CGMCC No.4509.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103222483A (en) * | 2013-01-07 | 2013-07-31 | 中国计量学院 | Use of Streptomyces sioyaensis metabolite in prevention and control of Botrytis cinerea |
| CN103222479A (en) * | 2013-01-07 | 2013-07-31 | 中国计量学院 | Use of Streptomyces sioyaensis metabolite in prevention and control of Colletotrichum lindemuthianum |
| CN103222484A (en) * | 2013-01-07 | 2013-07-31 | 中国计量学院 | Use of Streptomyces sioyaensis metabolite in prevention and control of Fusarium oxysporum. sp. cucumebrium Owen |
| CN117164542A (en) * | 2023-09-06 | 2023-12-05 | 南京中医药大学 | Compound with anti-tumor effect and preparation method thereof |
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| CN101040617A (en) * | 2007-01-25 | 2007-09-26 | 中国计量学院 | Application of Trichothec-9-en-4-o1,12,13-epoxy-,acetate,(4beta)-(8CI,9CI) in the preventing of tomato early blight or bean anthracnose |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103222483A (en) * | 2013-01-07 | 2013-07-31 | 中国计量学院 | Use of Streptomyces sioyaensis metabolite in prevention and control of Botrytis cinerea |
| CN103222479A (en) * | 2013-01-07 | 2013-07-31 | 中国计量学院 | Use of Streptomyces sioyaensis metabolite in prevention and control of Colletotrichum lindemuthianum |
| CN103222484A (en) * | 2013-01-07 | 2013-07-31 | 中国计量学院 | Use of Streptomyces sioyaensis metabolite in prevention and control of Fusarium oxysporum. sp. cucumebrium Owen |
| CN103222483B (en) * | 2013-01-07 | 2014-07-23 | 中国计量学院 | Use of Streptomyces sioyaensis metabolite in prevention and control of Botrytis cinerea |
| CN103222484B (en) * | 2013-01-07 | 2014-09-10 | 中国计量学院 | Use of Streptomyces sioyaensis metabolite in prevention and control of Fusarium oxysporum. sp. cucumebrium Owen |
| CN103222479B (en) * | 2013-01-07 | 2015-03-25 | 中国计量学院 | Use of Streptomyces sioyaensis metabolite in prevention and control of Colletotrichum lindemuthianum |
| CN117164542A (en) * | 2023-09-06 | 2023-12-05 | 南京中医药大学 | Compound with anti-tumor effect and preparation method thereof |
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