CN101979621A - Method for improving trichodermin content in fermentation liquor of Trichoderma harzianum strain L - Google Patents
Method for improving trichodermin content in fermentation liquor of Trichoderma harzianum strain L Download PDFInfo
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Abstract
The invention discloses a method for improving trichodermin content in fermentation liquor of Trichoderma harzianum strain L, and belongs to the technical field of microorganisms. The method comprises the following steps of: adding inactivated Fusarium oxysporum mycelium powder into a culture medium of the Trichoderma harzianum strain L for performing fermentation culture; performing centrifugation on the fermentation liquor; analyzing the trichodermin content in the fermentation liquor by adopting gas chromatography; and displaying that the concentration of trichodermin in the fermentation liquor after adding the inactivated Fusarium oxysporum mycelium powder is increased by several times. The invention provides a feasible method for improving the fermentation level for producing the trichodermin of the Trichoderma harzianum 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
Tire in the microbial fermentation and determine production cost and the bottleneck place of realizing suitability for industrialized production often.The optimization etc. of bacterial strain being carried out genetic modification, substratum and culture condition is to improve the common 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-dextran, acetyl glucosamine aldehydic acid, glycoprotein, 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 (Trichodermaharzianum) 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, is with a wide range of applications in preventing and treating crop pest.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 production 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 BL, DALY S and SINCLAIR J B.Antimycotic Compounds from the Plant PathogenRhizoctonia 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, the application in biological control for bigger performance trichoderma harzianum strain L and trichodermin, and 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, provide a kind of feasible method by the present invention for the fermentation level that improves trichodermin.
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, show that the concentration of trichodermin in the cucumber fusarium axysporum mycelium secondary fermentation liquid that adds deactivation has improved several times compared with the control.
The present invention improves the concentration of trichodermin in the fermented liquid by add the cucumber fusarium axysporum mycelium of deactivation in trichoderma harzianum strain L nutrient solution.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 at 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 is achieved through the following technical solutions:
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 is bought from Zhejiang Academy of Agricultural Science plant protection and institute of microbiology.Substratum is that (prescription is potato glucose (PD) liquid nutrient medium: the potato that takes by weighing the 200g peeling, be cut into piece and boil half an hour, use filtered through gauze then, add glucose 20g, supply water after dissolving to 1L), 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 is collected mycelium, at-53 ℃ through the centrifugal 10min of 5000r/min, 0.5mbar vacuum lyophilization 15h under the condition, the mycelium of deactivation is ground to Powdered under aseptic condition with mortar, standby;
(2) trichoderma harzianum strain L liquid fermenting
Trichoderma harzianum strain L be isolating endogenetic fungus from Chinese holly (stone one Jun, surname rising sun duckweed, the classification evaluation of the flat .1 strain of Yu Xiao holly fungal endophyte fungal bacterial strain and the biological and ecological methods to prevent plant disease, pests, and erosion effect of meta-bolites thereof research. 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 5000r/min, supernatant liquor 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 to add the cucumber fusarium axysporum mycelium of deactivation in trichoderma harzianum strain L nutrient solution, 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 is further described.
Embodiment 1:
The cultivation of cucumber fusarium axysporum: buy from Zhejiang Academy of Agricultural Science plant protection and institute of microbiology.Substratum is potato glucose liquid nutrient medium (PD), 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 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, standby;
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 liquor 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 breathing out trichodermin in time Trichoderma L fermented liquid has improved 4 times (being increased to 0.328g/L from 0.082g/L) compared with the control.
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 liquor 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 breathing out trichodermin in time Trichoderma L fermented liquid has improved 4.2 times (being increased to 0.344g/L from 0.082g/L) compared with the control.
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 liquor 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 has improved 4.25 times (being increased to 0.349g/L from 0.082g/L) compared with the control in the trichoderma harzianum strain L fermented liquid.
Embodiment 4:
The cultivation of botrytis cinerea: botrytis cinerea (Botrytis cinerea) is bought from Zhejiang Province's agricultural section's institute's plant protection and institute of microbiology.Substratum is potato glucose liquid nutrient medium (PD), 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 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, standby;
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 liquor 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 (4)
1. method that improves trichodermin content in trichoderma harzianum strain (Trichoderma harzianum) the L fermented liquid, it is characterized in that cucumber fusarium axysporum (Fusarium oxysporum) mycelium 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; 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 method for trichodermin content in the described raising trichoderma harzianum strain of the claim 1 L fermented liquid is characterized in that containing in the described substratum cucumber fusarium axysporum mycelium powder, peptone, Semen Maydis powder, glucose, wheat bran, the MnCl of deactivation
2, KH
2PO
4, MgSO
4
3. each component concentration of the described substratum of claim 2 (contained quality in every liter of substratum) is: the cucumber fusarium axysporum mycelium powder 60-90mg of deactivation, peptone 30g, Semen Maydis powder 10g, glucose 15g, wheat bran 5g, MnCl
20.01g, KH
2PO
40.1g, MgSO
40.05g.
4. the method for trichodermin content in the described raising trichoderma harzianum strain of the claim 1 L fermented liquid, the culturing process that it is characterized in that 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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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116235867A (en) * | 2022-11-30 | 2023-06-09 | 乐施康农业科技有限公司 | Pathogen for preventing and treating cotton wilt and preparation method thereof |
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|---|---|---|---|---|
| CN101037656A (en) * | 2007-01-25 | 2007-09-19 | 中国计量学院 | Horned holly fungal endophyte-harzianum L |
| 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 |
| CN101503659A (en) * | 2009-03-11 | 2009-08-12 | 中国农业大学 | Trichoderma harzianum strain and use thereof |
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Patent Citations (3)
| 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 |
| 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 |
| CN101503659A (en) * | 2009-03-11 | 2009-08-12 | 中国农业大学 | Trichoderma harzianum strain and use thereof |
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| 《Mycol. Res》 19941231 Scarselletti R.等 In vitro activity of 6-pentyl-1-pyrone,a metabolite of Trichoderma harzianum,in the inhibition of Rhizoctonia solani and Fusarium oxvsporum f, sp. lycopersici 1207-1209 1-4 , 2 * |
| 《PHYTOPATHOLOGY》 20100129 Ainhoa MartÃnez-Medina等 Trichoderma harzianum and Glomus intraradices Modify the Hormone Disruption Induced by Fusarium oxysporum Infection in Melon Plants 682-688 1-4 , 2 * |
| 《植物保护学报》 20100630 刘爱荣等 哈茨木霉对黄瓜尖孢镰刀菌的抑制作用和抗性相关基因表达 249-254 1-4 第37卷, 第3期 2 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116235867A (en) * | 2022-11-30 | 2023-06-09 | 乐施康农业科技有限公司 | Pathogen for preventing and treating cotton wilt and preparation method thereof |
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