CN100436575C - Heteromorphic Dactlella bacterial agent capable of killing nematode and application thereof - Google Patents
Heteromorphic Dactlella bacterial agent capable of killing nematode and application thereof Download PDFInfo
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- CN100436575C CN100436575C CNB2006100108505A CN200610010850A CN100436575C CN 100436575 C CN100436575 C CN 100436575C CN B2006100108505 A CNB2006100108505 A CN B2006100108505A CN 200610010850 A CN200610010850 A CN 200610010850A CN 100436575 C CN100436575 C CN 100436575C
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Abstract
The present invention relates to a heteromorphic dactlella bacterial agent capable of killing nematode and an application thereof, which belongs to the technical field of microbial pesticide. The production strain of the present invention is dactylell avarietas YMF1.00118, and is prepared by the conventional method, wherein the prescription of liquid fermentation culture medium comprises 0.1 percent of glucose, 0.1 percent of ammonia sulfate, 0.05 percent of magnesium sulfate, 0.001 percent of ferrous sulfate and 1000 ml of potato liquid at pH 6.5; the method of the nematode-killing active ingredient extracted from liquid fermentation products comprises fermentation culture is filtered, and supernatant is used as nematode-killing medicine liquid by using the ammonium sulfate for concentration. After the strain of the present invention is fermented by the liquid, the nematode-killing active ingredient is extracted form metabolic products to prepare microbe nematode-killing agent. The products of the present invention have the obvious advantage of high toxicity for full-tooth renatured nematode and caenorhabditis elegans, and have good latent application perspective.
Description
Technical field:
The present invention relates to a kind of heterophragma and refer to spore (Dactylella varietas) crude enzyme liquid and application, belong to biological pesticide technical field with nematode killing function.
Background technology:
Plant nematode is the Plant diseases that generally takes place in a kind of world wide, and only the known kind of root knot nematode reaches kind more than 70, endangers 3000 various plants, and cause damage huge every year.The annual crop of being caused by plant nematode in the whole world is with a toll of 78,000,000,000 dollars (Barker, 1998) according to estimates.Calendar year 2001, the loss that causes because of nematode harm of China crop was up to 23,300,000,000 yuan.Pine wood nematode is called as smokeless hill fire, in Jiangsu, partly the area is serious Zhejiang, Guangdong, Shandong, Anhui, Hubei, Shanghai, Taiwan, Hong Kong etc. that the trend of stretching in the oriented whole nation takes place.More seriously, cause the crop root disease seriously to take place because root knot nematode is caused the crop wound after infecting.
At present to control of nematode still based on chemical nematocides.Invent in the eighties of last century chemical nematocides of the forties, ensureing agriculture production for a long time. prevent and treat and brought into play vital role in the oxyuriasis.But along with progress of science and technology, find that many chemical nematocidess all have side effect, not only develop immunity to drugs, more cause environmental pollution, be detrimental to health, have many disabled or be about to forbidding in succession.Banned use of monobromethane as the U.S., methylene bromide can only use 2005.The chemistry nematocides when ensureing agriculture production, has brought serious negative impact for human health and environment at the control eelworm harm, and exploitation high-efficiency low-toxicity nematocides and biological nematocides are extremely urgent.
Every referring to that spore is the very important biocontrol fungi resource of a class, all be the pathogenic fungi of very important insect and nematode much wherein, can effectively be used to prevent and treat multiple harmful insect and nematode, in the biological control of the cause of disease insect of plant and nematode, play important effect.At present, increasing about the report of plant nematode biological control, yet without any referring to that about heterophragma spore kills the report of insect and nematode.
Summary of the invention:
The objective of the invention is to refer to the research of spore, the exploitation biological mematocide by the heterophragma that also can effectively decompose the nematode body wall structure to strain generation extracellular enzyme.
The strain that the present invention screens refers to spore (Dactylellavarietas), YMF1.00118 to the heterophragma that plant nematode has fine toxic action.Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: China. Beijing. the Zhong Guan-cun; Preservation date: on October 31st, 2005; The numbering CGMCC No.1521 that preservation is registered on the books.
Heterophragma refer to spore (Dactylella varietas YMF1.00118) is this laboratory isolating Nematophagous fungi from pedotheque, and main morphological specificity is:
This bacterial strain is grown slow on PDA and CMA flat board, and the two weeks colony diameter has only 2-3cm, hyphae colorless, do not separate, branch is not easy to produce spore, and sporophore uprightly has separation, some has branch, the top gives birth to single conidium (seeing two once in a while), and spore is shaped as typical fusiform (6-10 μ m-40-60 μ m), separates to change big (2-8), 5-8 separation is in the majority, and the mycelia specialization forms the mycelia circle of three-dimensional bacterium net and cover strand oligotrophic the time.
The present invention prepares according to a conventional method.After soon heterophragma will refer to the spore enlarged culturing, extract nematicide effective constituent, measure it brings back to life nematode (Panagrellus redivivus) and Caenorhabditis elegans nematode (Caenorhabditiselegans) to full-depth tooth insecticidal effect.
The present invention is achieved in that
YMF1.00118 strain culturing method:
1. the test tube kind is cultivated
Culture medium prescription is a modified form PDA substratum: 1% glucose; 1000ml murphy juice (the 100g potato adds water 1200ml and boils filtration), 1.8-2% agar.The YMF1.00118 mycelium is inoculated on the substratum, cultivated 7～10 days down, obtain the test tube kind for 20-25 ℃.
2. culture dish enlarged culturing
Adopt aforementioned 1 modified form PDA substratum, diameter is 9 centimetres of culture dish, behind the inoculation YMF1.00118, cultivates 7～10 days down in 20-25 ℃ after film seals with sealing, and obtains to be used for the bacterial classification of fluid enlargement culture.
3. fluid enlargement culture (produce enzyme cultivate)
It is composed as follows to produce enzyme substratum (PL-4): 0.1% glucose, and 0.1% ammonium sulfate, 0.05% sal epsom, 0.001% ferrous sulfate, 1000ml murphy juice (the 100g potato adds water 1200ml and boils filtration), pH 6.5.Each 250ml triangular flask packing 60ml nutrient solution, and add a spot of nematode as inductor, sterilized 20 minutes for 121 ℃, insert the bacterium piece, 26 ℃ of shaking tables (200rpm) were cultivated 6 days.
The extraction of eelworm-killing activity composition:
Heterophragma refers to spore mycelium inoculation PDA substratum, incubated at room temperature 10 days, and the PL-4 substratum of picking inoculated by hypha block sterilization, 26 ℃, the 200rpm shaking table was cultivated 6 days.Collect supernatant liquor (2 liters), add ammonium sulfate (85% saturation ratio) and precipitate, resolution of precipitate is in an amount of phosphate buffered saline buffer.Crude enzyme liquid carries out purifying with cationic exchange coloum after handling through the dialysis desalination, collect respectively and penetrate peak and elution peak, biological activity determination shows most of protease activity at elution peak, collects elution peak and handles with distill water dialysis, and it is pure that electrophoretic analysis has reached electrophoresis.Lyophilize is the sample freeze-drying and be dissolved in the distilled water of 2ml-20 ℃ of preservations.Concentrating later sample through ultra-filtration membrane can use as the desinsection soup.
The eelworm-killing activity test:
1, preparation test with medicament and contrast with medicament
1) cultivates the YMF1.00118 bacterial strain by aforementioned liquids enlarged culturing method, by the method preparation test with medicament of aforementioned extraction eelworm-killing activity composition.
2) preparation contrast with medicament
Contrast 1: aforementioned 1 preparation test with medicament method preparation contrast with medicament, will prepare the reagent agent heated and boiled after 20 minutes in contrast;
Contrast 2: in order to prove the nematicide effective ingredient is albumen, will prepare reagent agent with after the proteinase inhibitor processing in contrast.
2, nematode is used in the preparation test
Full-depth tooth is brought back to life nematode (P.redivivus) and Caenorhabditis elegans nematode (C.elegans) is inoculated in respectively on the medium oatmeal of sterilization, cultivated 6-10 days down, preserve standby in 4 ℃ of refrigerators in 28 ℃.Required nematode is washed out with the graceful funnel method of shellfish, place in the 5ml centrifuge tube, add the washing of 5ml sterilized water, instantaneous centrifugal, abandon supernatant, repeat 5 times and obtain clean for the examination nematode.Is that content is the nematode suspension of 15/μ l with sterilized water with the nematode dilution.
3, test method
1) test of pesticide effectiveness method
Get experimental drug agent 200ul in the 1.5ml centrifuge tube, add 60 of living nematodes, centrifuge tube keeps flat, and places 25 ℃, observes at microscopically respectively at 12 hours and 24 hours, and calculates the mortality ratio of full-depth tooth resurrection nematode and Caenorhabditis elegans nematode respectively.The method of identifying nematode death is: add 1-5 and drip 5%NaCl solution in centrifuge tube, observe after 2 minutes, dead worm is stiff, and the worm that lives is then curled or twisting.
Carry out control experiment with 2 kinds of contrast medicaments respectively.
The test establish three parallel, twice repetition.
2) nematocidal effect of purification of samples and to the decomposition of nematode body wall: experimental technique is the same, with the sample of the observation by light microscope purifying Decomposition to the nematode body wall.
4, test-results
Table 1:YMF1.00118 brings back to life the insecticidal effect of nematode (Panagrellus redivivus) to full-depth tooth
Table 2:YMF1.00118 is to the insecticidal effect of Caenorhabditis elegans nematode (C.elegans)
The result shows, the test with medicament of extracting from YMF1.00118 strain liquid tunning is brought back to life nematode and the equal tool of Caenorhabditis elegans nematode nematicide effect preferably to full-depth tooth.Proteolytic enzyme with 10 times of spissated thick enzyme samples and purifying is handled nematode as soup, full-depth tooth is brought back to life nematode and 100% death of Caenorhabditis elegans nematode after 24 hours, and most of nematode be decomposed fully (table 1 and table 2), experimental result shows that the eelworm-killing activity of the sample of purifying is lower than thick concentrating sample simultaneously, illustrated that multiple composition has participated in nematicide process, so the desinsection soup is selected thick concentrating sample for use.
From handling the variation of nematode, experienced from the part body wall and decomposed (accompanying drawing is to show) process to the whole degradeds of polypide, illustrate that active ingredient is mainly the enzyme that decomposes nematode.And will be with batch test with medicament of extracting through boiling and inhibitor is handled, cause protein denaturation after, its eelworm-killing activity is very little, and dissolution phenomena do not appear in the nematode body wall, this has proved that also heterophragma refers to that spore is mainly enzyme to the active ingredient of nematode.
Heterophragma of the present invention refers to that spore YMF1.00118 is a kind of Nematophagous fungi, mainly to act on composition be proteolytic enzyme to the excretory extracellular enzyme in the used substratum of the present invention, full-depth tooth resurrection nematode and Caenorhabditis elegans nematode all there are good insecticidal effect, have possessed the good application development prospect.
Description of drawings:
Accompanying drawing shows that heterophragma refers to that the extracellular protease of spore brings back to life the Degradation of nematode body wall to full-depth tooth.
Embodiment:
Below be embodiments of the invention, but content of the present invention is not limited thereto.
Embodiment one:
To improvement PDA inclined-plane, culture medium prescription is: 1% glucose with the YMF1.00118 inoculation; The 100g potato boils into juice, 1.8-2% agar; The pH nature.The YMF1.00118 mycelium is inoculated on the substratum, cultivated 7～10 days down, obtain the test tube kind for 20-25 ℃.
The test tube kind is inoculated into (every bottled 60ml) in the 250ml triangular flask liquid nutrient medium, and the liquid culture based formulas is: 0.1% glucose, 0.1% ammonium sulfate, 0.05% sal epsom, the 100g potato boils into juice, 0.001% ferrous sulfate, water replenishes volume to 1L, and pH 6.5.Each 250ml triangular flask packing 60ml nutrient solution, and add a spot of nematode as inductor, sterilized 20 minutes for 121 ℃, insert the bacterium piece, 26 ℃ of shaking tables (200rpm) were cultivated 6 days.
Heterophragma refers to spore mycelium inoculation PDA substratum, incubated at room temperature 10 days, and the PL-4 substratum of picking inoculated by hypha block sterilization, 26 ℃, the 200rpm shaking table was cultivated 6 days.Collect supernatant liquor (2 liters), add ammonium sulfate (85% saturation ratio) and precipitate, resolution of precipitate is in an amount of phosphate buffered saline buffer.Crude enzyme liquid carries out purifying with cationic exchange coloum after handling through the dialysis desalination, collect respectively and penetrate peak and elution peak, biological activity determination shows most of protease activity at elution peak, collects elution peak and handles with distill water dialysis, and it is pure that electrophoretic analysis has reached electrophoresis.Lyophilize is the sample freeze-drying and be dissolved in the distilled water of 2ml-20 ℃ of preservations.Concentrating later sample through ultra-filtration membrane can use as the desinsection soup.
Embodiment two:
Substantially with embodiment one, difference is the liquid culture based formulas, and its prescription is: 0.1% glucose, and 0.1% ammonium sulfate, 0.05% sal epsom, the 100g potato boils into juice, 0.001% ferrous sulfate, 0.1% peptone, water replenish volume to 1L, and pH 6.5.
Claims (2)
1, a kind of heterophragma with nematode killing function refers to the spore crude enzyme liquid, and the extraction by the cultivation of test tube kind, culture dish enlarged culturing, fluid enlargement culture, eelworm-killing activity composition obtains, and it is characterized in that:
A. heterophragma refers to that the production bacterial strain of spore crude enzyme liquid is Dactylella varietas YMF1.00118, and preserving number is CGMCC No.1521;
B. the test tube kind is cultivated: culture medium prescription is a modified form PDA substratum: 1% glucose; The 100g potato adds the 1000ml murphy juice after water 1200ml boils filtration, 1.8-2% agar; The YMF1.00118 mycelium is inoculated on the substratum, cultivated 7～10 days down, obtain the test tube kind for 20-25 ℃;
C. culture dish enlarged culturing: adopt the described modified form PDA of b substratum, diameter is 9 centimetres of culture dish, behind the inoculation YMF1.00118, seals the back and cultivates 7～10 days down in 20-25 ℃ with sealing film, and acquisition is used for the bacterial classification of fluid enlargement culture;
D. fluid enlargement culture: produce consisting of of enzyme substratum: 0.1% glucose, 0.1% ammonium sulfate, 0.05% sal epsom, 0.001% ferrous sulfate, 100g potato add the 1000ml murphy juice after water 1200ml boils filtration, pH6.5; Each 250ml triangular flask packing 60ml nutrient solution, and add a spot of nematode as inductor, 121 ℃ of sterilizations 20 minutes insert the bacterial classification that the c step is cultivated, and cultivate 6 days in 26 ℃ rotating speeds are the shaking table of 200rpm.
2, the described heterophragma of claim 1 refers to the application of spore crude enzyme liquid, it is characterized in that the biological prevention and control agent of this crude enzyme liquid as plant nematode.
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| CNB2006100108505A CN100436575C (en) | 2006-04-26 | 2006-04-26 | Heteromorphic Dactlella bacterial agent capable of killing nematode and application thereof |
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| CN100436575C true CN100436575C (en) | 2008-11-26 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6168947B1 (en) * | 1999-02-11 | 2001-01-02 | Food Industry Research And Development Institute | Nematophagous fungus Esteya vermicola |
| CN1462583A (en) * | 2003-05-21 | 2003-12-24 | 云南大学 | Method of producing biology agent for killing thread worm |
| WO2004077944A1 (en) * | 2003-03-04 | 2004-09-16 | Japan Applied Microbiology Research Institute Ltd. | Nematocide |
| CN1673348A (en) * | 2005-03-16 | 2005-09-28 | 云南大学 | Nematode-eating fungus with nematode-killing function, preparing method and use thereof |
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- 2006-04-26 CN CNB2006100108505A patent/CN100436575C/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6168947B1 (en) * | 1999-02-11 | 2001-01-02 | Food Industry Research And Development Institute | Nematophagous fungus Esteya vermicola |
| WO2004077944A1 (en) * | 2003-03-04 | 2004-09-16 | Japan Applied Microbiology Research Institute Ltd. | Nematocide |
| CN1462583A (en) * | 2003-05-21 | 2003-12-24 | 云南大学 | Method of producing biology agent for killing thread worm |
| CN1673348A (en) * | 2005-03-16 | 2005-09-28 | 云南大学 | Nematode-eating fungus with nematode-killing function, preparing method and use thereof |
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