CN101979619B - Application of Fusarium oxysporiumf.sp.cucumeris in improving level of producing trichodermin of trichoderma harziarum strain L - Google Patents
Application of Fusarium oxysporiumf.sp.cucumeris in improving level of producing trichodermin of trichoderma harziarum strain L Download PDFInfo
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- CN101979619B CN101979619B CN2010105276414A CN201010527641A CN101979619B CN 101979619 B CN101979619 B CN 101979619B CN 2010105276414 A CN2010105276414 A CN 2010105276414A CN 201010527641 A CN201010527641 A CN 201010527641A CN 101979619 B CN101979619 B CN 101979619B
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Abstract
The invention discloses application of Fusarium oxysporiumf.sp.cucumeris in improving level of producing trichodermin of a trichoderma harziarum strain L, and belongs to the technical field of microorganisms. The method comprises the following steps of: adding inactivated Fusarium oxysporiumf.sp.cucumeris mycelium powder into a culture medium for the trichoderma harziarum strain L for fermentation culture; centrifuging fermentation liquor; and analyzing the content of the trichodermin in the fermentation liquor by adopting a gas chromatogram, wherein the concentration of the trichodermin in the fermentation liquor after the inactivated Fusarium oxysporiumf.sp.cucumeris mycelium powder is added is improved by several times than that of a contrast. The invention provides a feasible method for improving the fermentation level of producing the trichodermin of the trichoderma harziarum strain L.
Description
Technical field
The present invention relates to microbial technology field, particularly relate to the method that improves gliotoxin production level of harziarum strain.
Background technology
The bottleneck place of tiring in the microbial fermentation and determining production cost often and realizing suitability for industrialized production.The optimization etc. of bacterial strain being carried out genetic modification, substratum and culture condition is to improve the domestic method of producing plain level.In recent years, fungal elicitor is used widely in the research of culture plant cell and microbial fermentation production secondary metabolite.Fungal elicitor mainly is structure of cell surface or the secretory product of fungi; They can be mycelium, mycelium degraded product, fermented liquid, fungal secretion of fungi etc., mainly comprise: polysaccharide, chitosan, β-1-3-VISOSE, acetyl glucosamine aldehydic acid, gp, protein, small peptide, unsaturated fatty acids etc.The application report of producing active secondary metabolite at microbial fermentation about fungal elicitor seldom.Study group's separation screening from Chinese holly produces the active endogenetic fungus-trichoderma harzianum (Trichoderma harzianum) of trichodermin (Trichodermin) to a strain, names to be trichoderma harzianum strain (Trichoderma harzianum) L.This bacterial strain is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preservation date on August 14th, 2006, preservation registration number CGMCC No.1780.Trichodermin has very strong restraining effect to the multiple kinds of crops pathogenic fungi, in preventing and treating crop pest, is with a wide range of applications.It is lower often to produce plain level from the isolating wild type strain of nature, and throughput is low.
The document of external relevant trichoderma harzianum fermentative prodn trichodermin is very few; The unit that the trichoderma harzianum strain Th008 of reports such as BERTAGNOLLI produces trichodermin is 0.324 μ g/mL (BERTAGNOLLI B L; DALY S and SINCLAIR J B.Antimycotic Compounds from the Plant Pathogen Rhizoctonia solani and its Antagonist Trichoderma harzianum.J.Phytopathology; 1998,146,131-135.).The existing product of the trichoderma harzianum strain L trichodermin unit that study group is separated to is about 80mg/L, and for bigger performance trichoderma harzianum strain L and the application of trichodermin in biological control, improving the trichodermin productive rate is gordian technique.On the research basis in early stage, find to add microorganism induction and can improve the trichodermin productive rate effectively, for the fermentation level that improves trichodermin a kind of feasible method is provided through the present invention.
Summary of the invention
Improve trichoderma harzianum strain L in research and produce in the test of plain level, biological activity determination shows that the mycelial adding of cucumber fusarium axysporum (Fusarium oxysporum) of deactivation can improve the fungistatic effect of trichoderma harzianum strain L fermented liquid.Adopt the content of trichodermin in the gas chromatographic analysis fermented liquid, the concentration of trichodermin is compared with contrast and has been improved several times in the cucumber fusarium axysporum mycelium secondary fermentation liquid of demonstration interpolation deactivation.
The present invention improves the concentration of trichodermin in the fermented liquid through in trichoderma harzianum strain L nutrient solution, adding the cucumber fusarium axysporum mycelium of deactivation.This invention provides a kind of reliable method for the plain level of the product of trichoderma harzianum strain L.
The objective of the invention is to produce the low deficiency of plain level, a kind of method that it produces plain level that improves is provided to wild type strain trichoderma harzianum strain L; Another object of the present invention provides the method for utilizing this bacterial strain meta-bolites trichodermin to prepare microbial pesticide.
The object of the invention realizes through following technical scheme:
A kind of method that improves trichodermin content in the trichoderma harzianum strain L fermented liquid is carried out according to the following steps:
(1) cultivation of cucumber fusarium axysporum
Cucumber fusarium axysporum, plant protection and institute of microbiology buy from the Zhejiang Academy of Agricultural Science.Substratum is that (prescription is potato glucose (PD) liquid nutrient medium: take by weighing the yam of 200g peeling, be cut into piece and boil half a hour, use filtered through gauze then; Add glucose 20g, supply water to 1L after dissolving), the bottled PD liquid nutrient medium of 300mL triangle 60mL; In the culturing bottle of sterilization, put 26 ℃～28 ℃ shaking tables, 200r/min with a little cucumber fusarium axysporum mycelia of inoculation hook picking; Shaking culture 6 days, fermented liquid are collected mycelium through the centrifugal 10min of 5000r/min; Vacuum lyophilization 15h under-53 ℃, 0.5mbar condition, the mycelium of deactivation is ground to Powdered under aseptic condition with mortar, subsequent use;
(2) trichoderma harzianum strain L liquid fermenting
Trichoderma harzianum strain L is isolating endogenetic fungus stone one Jun from Chinese holly, surname rising sun duckweed, the biological and ecological methods to prevent plant disease, pests, and erosion effect research of the classification evaluation of the flat .1 strain holly fungal endophyte fungal bacterial strain of Yu Xiao and meta-bolites thereof. Plant Pathology, 2009,39 (4): 362-367).Fermentative medium formula: its component and content (contained quality in every liter of substratum) are cucumber fusarium axysporum mycelium powder 60mg, peptone 30g, Semen Maydis powder 10g, glucose 15g, wheat bran 5g, MnCl
20.01g, KH
2PO
40.1g, MgSO
40.05g; Do not add the cucumber fusarium axysporum mycelium powder of deactivation in the contrast, the same fermentative medium formula of all the other components and content; The bottled fermention medium 60mL of 300mL triangle draws 1mL trichoderma harzianum strain L spore suspension (1 * 10 with aseptic suction pipe
6Individual/mL) in the sterilization culturing bottle in, put 28 ℃ of shaking tables, 180r/min, shaking culture 96h, fermented liquid is through the centrifugal 10min of 5000rmin, supernatant is used for the trichodermin Determination on content;
(3) detection of trichodermin
Method: adopt gas chromatography determination, chromatographic column HP-5 fused-silica capillary column (with 5% phenyl methyl siloxanes is stationary liquid, 30m * 320 μ m * 0.25 μ m); Detector: hydrogen flame ionization detector (FID); 270 ℃ of detector temperatures; 250 ℃ of injector temperatures; Column temperature: temperature programming, 100 ℃ of initial temperatures keep 5min, with 10 ℃ of min
-1Be warming up to 260 ℃ and keep 5min; Carrier gas: nitrogen; Flow velocity 1.5mLmin
-1
Beneficial effect of the present invention:
The one, the present invention passes through in trichoderma harzianum strain L nutrient solution, to add the cucumber fusarium axysporum mycelium of deactivation, improves the concentration of trichodermin in the fermented liquid, and the plain level of this product for trichoderma harzianum strain L provides a kind of reliable method;
The 2nd, the invention provides the activeconstituents trichodermin of the microbial pesticide of preparation control crop fungal disease, for the exploitation of disinfectant use in agriculture has increased new approach.
Embodiment
Below in conjunction with embodiment the present invention being improved the method that trichodermin produces plain level further describes.
Embodiment 1:
The cultivation of cucumber fusarium axysporum: plant protection and institute of microbiology buy from the Zhejiang Academy of Agricultural Science.Substratum is potato glucose liquid nutrient medium (PD), and the bottled PD liquid nutrient medium of 300mL triangle 60mL is with inoculating a little cucumber fusarium axysporum mycelia of hook picking in the culturing bottle of sterilization; Put 26 ℃～28 ℃ shaking tables, 200r/min, shaking culture 6 days; Fermented liquid is through the centrifugal 10min of 5000r/min, collects mycelium, vacuum lyophilization 15h under-53 ℃, 0.5mbar condition; The mycelium of deactivation is ground to Powdered under aseptic condition with mortar, subsequent use;
Trichoderma harzianum strain L liquid fermenting: trichoderma harzianum strain L is an isolating endogenetic fungus from Chinese holly; This bacterial strain is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation; Preservation date on August 14th, 2006, preservation registration number CGMCC No.1780.Fermentative medium formula: its component and content (contained quality in every liter of substratum) are cucumber fusarium axysporum mycelium powder 60mg, peptone 30g, Semen Maydis powder 10g, glucose 15g, wheat bran 5g, MnCl
20.01g, KH
2PO
40.1g, MgSO
40.05g; Do not add the cucumber fusarium axysporum mycelium powder of deactivation in the contrast, the same fermentative medium formula of all the other components and content; The bottled fermention medium 60mL of 300mL triangle draws 1mL trichoderma harzianum strain L spore suspension (1 * 10 with aseptic suction pipe
6Individual/mL) in the sterilization culturing bottle in, put 28 ℃ of shaking tables, 180r/min, shaking culture 96h, fermented liquid is through the centrifugal 10min of 5000r/min, supernatant is used for the trichodermin Determination on content;
The detection of trichodermin: adopt gas chromatography determination, chromatographic column HP-5 fused-silica capillary column (with 5% phenyl methyl siloxanes is stationary liquid, 30m * 320 μ m * 0.25 μ m); Detector: hydrogen flame ionization detector (FID); 270 ℃ of detector temperatures; 250 ℃ of injector temperatures; Column temperature: temperature programming, 100 ℃ of initial temperatures keep 5min, with 10 ℃ of min
-1Be warming up to 260 ℃ and keep 5min; Carrier gas: nitrogen; Flow velocity 1.5mLmin
-1
The result shows: it is last to add the cucumber fusarium axysporum mycelium powder, and the content of trichodermin is compared with contrast and improved 4 times (being increased to 0.328g/L from 0.082g/L) in time Trichoderma L fermented liquid of Kazakhstan.
Embodiment 2:
The cultivation of cucumber fusarium axysporum: with embodiment 1;
Trichoderma harzianum strain L liquid fermenting: trichoderma harzianum strain L is an isolating endogenetic fungus from Chinese holly; Fermentative medium formula: its component and content (contained quality in every liter of substratum) are cucumber fusarium axysporum mycelium powder 90mg, peptone 30g, Semen Maydis powder 10g, glucose 15g, wheat bran 5g, MnCl
20.01g, KH
2PO
40.1g, MgSO
40.05g; Do not add the cucumber fusarium axysporum mycelium powder of deactivation in the contrast, the same fermentative medium formula of all the other components and content; The bottled fermention medium 60mL of 300mL triangle draws 1mL trichoderma harzianum strain L spore suspension (1 * 10 with aseptic suction pipe
6Individual/mL) in the sterilization culturing bottle in, put 28 ℃ of shaking tables, 180r/min, shaking culture 96h, fermented liquid is through the centrifugal 10min of 5000r/min, supernatant is used for the trichodermin Determination on content;
The detection of trichodermin: with embodiment 1;
The result shows: it is last to add the cucumber fusarium axysporum mycelium powder, and the content of trichodermin is compared with contrast and improved 4.2 times (being increased to 0.344g/L from 0.082g/L) in time Trichoderma L fermented liquid of Kazakhstan.
Embodiment 3:
The cultivation of cucumber fusarium axysporum: with embodiment 1;
Trichoderma harzianum strain L liquid fermenting: trichoderma harzianum strain L is an isolating endogenetic fungus from Chinese holly; Fermentative medium formula: its component and content (contained quality in every liter of substratum) are cucumber fusarium axysporum mycelium powder 90mg (adding after 24 hours at the trichoderma harzianum liquid fermenting), peptone 30g, Semen Maydis powder 10g, glucose 15g, wheat bran 5g, MnCl
20.01g, KH
2PO
40.1g, MgSO
40.05g; Do not add the cucumber fusarium axysporum mycelium powder of deactivation in the contrast, the same fermentative medium formula of all the other components and content; The bottled fermention medium 60mL of 300mL triangle draws 1mL trichoderma harzianum strain L spore suspension (1 * 10 with aseptic suction pipe
6Individual/mL) in the sterilization culturing bottle in, put 28 ℃ of shaking tables, 180r/min, shaking culture 108h, fermented liquid is through the centrifugal 10min of 5000r/min, supernatant is used for the trichodermin Determination on content;
The detection of trichodermin: with embodiment 1;
The result shows: after adding the cucumber fusarium axysporum mycelium, the content of trichodermin is compared with contrast and has been improved 4.25 times (being increased to 0.349g/L from 0.082g/L) in the trichoderma harzianum strain L fermented liquid.
Embodiment 4:
The cultivation of botrytis cinerea: botrytis cinerea (Botrytis cinerea) is agricultural section's institute's plant protection and institute of microbiology's purchase from Zhejiang Province.Substratum is potato glucose liquid nutrient medium (PD), and the bottled PD liquid nutrient medium of 300mL triangle 60mL is with inoculating a little cucumber fusarium axysporum mycelia of hook picking in the culturing bottle of sterilization; Put 26 ℃～28 ℃ shaking tables, 200r/min, shaking culture 6 days; Fermented liquid is through the centrifugal 10min of 5000r/min, collects mycelium, vacuum lyophilization 15h under-53 ℃, 0.5mbar condition; The mycelium of deactivation is ground to Powdered under aseptic condition with mortar, subsequent use;
Trichoderma harzianum strain L liquid fermenting: trichoderma harzianum strain L is an isolating endogenetic fungus from Chinese holly; Fermentative medium formula: its component and content (contained quality in every liter of substratum) are botrytis cinerea mycelium powder 60mg, peptone 30g, Semen Maydis powder 10g, glucose 15g, wheat bran 5g, MnCl
20.01g, KH
2PO
40.1g, MgSO
40.05g; Do not add botrytis cinerea mycelium powder in the contrast, the same fermentative medium formula of all the other components and content; The bottled fermention medium 60mL of 300mL triangle draws 1mL trichoderma harzianum strain L spore suspension (1 * 10 with aseptic suction pipe
6Individual/mL) in the sterilization culturing bottle in, put 28 ℃ of shaking tables, 180r/min, shaking culture 96h, fermented liquid is through the centrifugal 10min of 5000r/min, supernatant is used for the trichodermin Determination on content;
The detection of trichodermin: adopt gas chromatography determination, chromatographic column HP-5 fused-silica capillary column (with 5% phenyl methyl siloxanes is stationary liquid, 30m * 320 μ m * 0.25 μ m); Detector: hydrogen flame ionization detector (FID); 270 ℃ of detector temperatures; 250 ℃ of injector temperatures; Column temperature: temperature programming, 100 ℃ of initial temperatures keep 5min, with 10 ℃ of min
-1Be warming up to 260 ℃ and keep 5min; Carrier gas: nitrogen; Flow velocity 1.5mLmin
-1
The result shows: it is last to add the botrytis cinerea mycelium powder, and the content of trichodermin (0.0825g/L liter) is compared basic not raising in the trichoderma harzianum strain L fermented liquid with contrast (0.0820g/L liter).
Claims (2)
1. the application of cucumber fusarium axysporum (Fusarium oxysporum) mycelium in improving trichoderma harzianum strain (Trichoderma harzianum) L production trichodermin level; It is characterized in that the cucumber fusarium axysporum mycelium powder of deactivation is joined in the substratum of trichoderma harzianum strain L; Trichoderma harzianum strain L is carried out cultivation and fermentation, carry out centrifugal to fermented liquid then; Wherein trichoderma harzianum strain L is an isolating endogenetic fungus from Chinese holly, and this bacterial strain is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preservation date on August 14th, 2006, preservation registration number CGMCC No.1780.
2. the described application of claim 1, it is characterized in that the culturing process of cucumber fusarium axysporum is: substratum is the potato glucose liquid nutrient medium, with inoculating a little cucumber fusarium axysporum mycelia of hook picking in the culturing bottle of sterilization, the shaking table shaking culture; Fermented liquid is centrifugal then, collects mycelium, and vacuum lyophilization is ground to the mycelium of deactivation Powdered under aseptic condition with mortar.
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| CN101037656A (en) * | 2007-01-25 | 2007-09-19 | 中国计量学院 | Horned holly fungal endophyte-harzianum L |
| CN101041840A (en) * | 2007-01-25 | 2007-09-26 | 中国计量学院 | Preparation method of sesquiterpenoids Trichothec-9-en-4-o1,12,13-epoxy-,acetate,(4beta)-(8CI,9CI) |
| CN101270373A (en) * | 2008-05-19 | 2008-09-24 | 中国计量学院 | Method for improving gliotoxin production level of harziarum strain |
| CN101275152A (en) * | 2008-05-19 | 2008-10-01 | 中国计量学院 | Application of rhizoctonia solani mycelium to improving trichodermin in trichoderma harzianum |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101037656A (en) * | 2007-01-25 | 2007-09-19 | 中国计量学院 | Horned holly fungal endophyte-harzianum L |
| CN101041840A (en) * | 2007-01-25 | 2007-09-26 | 中国计量学院 | Preparation method of sesquiterpenoids Trichothec-9-en-4-o1,12,13-epoxy-,acetate,(4beta)-(8CI,9CI) |
| CN101270373A (en) * | 2008-05-19 | 2008-09-24 | 中国计量学院 | Method for improving gliotoxin production level of harziarum strain |
| CN101275152A (en) * | 2008-05-19 | 2008-10-01 | 中国计量学院 | Application of rhizoctonia solani mycelium to improving trichodermin in trichoderma harzianum |
Non-Patent Citations (1)
| Title |
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| 石一珺等.1株枸骨内生真菌菌株的分类鉴定及其代谢产物的生防作用研究.《植物病理学报》.2009,第39卷(第4期),362-367. * |
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