CN100401899C - Bacterial agent capable of killing nematode and application thereof - Google Patents
Bacterial agent capable of killing nematode and application thereof Download PDFInfo
- Publication number
- CN100401899C CN100401899C CNB2006100106834A CN200610010683A CN100401899C CN 100401899 C CN100401899 C CN 100401899C CN B2006100106834 A CNB2006100106834 A CN B2006100106834A CN 200610010683 A CN200610010683 A CN 200610010683A CN 100401899 C CN100401899 C CN 100401899C
- Authority
- CN
- China
- Prior art keywords
- nematode
- strain
- present
- test tube
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The present invention relates to a bacterial agent capable of killing nematode and an application thereof, which belongs to the technical field of microbial pesticide. The bacterial agent of the present invention is obtained by production strain through the strain culture in a test tube, fluid enlargement culture and nematode-killing active component extraction, and the production strain of the present invention is stenotrophomonas maltophilia G6; the strain G6 is preserved at the common microorganisms center of the CCCCM, the preservation date of the present invention is the 31th of October in 2005, and the preservation booked number is CGMCC No. 1522. Though enzyme production and nematode body wall decomposition, the main action mechanism of the strain G6 provides the obvious degermation for nematode, particular pine wood nematode so that microbe nematicide can be prepared. The product of the present invention has the obvious advantage of high toxicity for pine wood nematode, and has good potential application perspective.
Description
Technical field:
The present invention relates to a kind of microbial inoculum and application thereof, the microorganism belonging to genus technical field of pesticide with nematocide function.
Background technology:
Plant nematode is the plant disease that generally takes place in a kind of world wide, and only the known kind of root-knot nematode reaches kind more than 70,3000 various plants that cause harm, and the loss that causes every year is huge.According to FAO's statistics, cause damage the whole world up to 1,000 hundred million dollar because of nematode every year.In China, main economic crops such as nematode main harm tobacco, flowers, vegetables, cotton, soybean, peanut, pseudo-ginseng, American Ginseng become the critical limitation factor that develops these crops.According to the incomplete statistics of national tobacco disease pest control information centre, only Yunnan Province's onset area just reaches 2.7 ten thousand public affairs and inclines, 1.2 hundred million yuan of direct economic losses, and indirect loss is even more serious.The Heilongjiang Province only the soybean Cyst nematode cause every year with a toll of 800,000,000 yuan more than.Pine wood nematode is called as smokeless forest 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.In Shandong, vegetables main producing region such as Guangdong, Hainan and Yunnan, root knot nematode is in rising trend.More seriously, cause the crop root disease seriously to take place owing to after root-knot nematode infects, cause the crop wound.The pseudo-ginseng root-rot in Yunnan is exactly an exemplary.The chemistry nematocide has been brought into play important function in nematoda control since the eighties of last century discovery forties.But along with progress of science and technology, find that many chemical nematocides all have side effect, cause environmental pollution, be detrimental to health.Have many disabled or be about to forbidding in succession.Particularly plant nematode betides crop root, because the particularity of soil environment, must heavy dose uses and could guarantee its preventive effect.And heavy dose of chemical pesticide is very serious to groundwater contamination.Therefore, research and development high-efficiency low-toxicity low-residual nematocide has been the important topic in the agricultural sustainable development.
By literature search, do not see the open report identical with the present invention.
Summary of the invention:
The objective of the invention is to produce the stenotrophomonas maltophilia research that enzyme decomposes the nematode function, develop biological nematocide by a strain is had.
The strain that the present invention screens has stenotrophomonas maltophilia (stenotrophomonas maltophilia) G6 of fine insecticidal function to plant nematode, the G6 bacterial strain has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date: on October 31st, 2005; The numbering CGMCC No:1522 that preservation is registered on the books.
Stenotrophomonas maltiphilia G6 strain morphology of the present invention is characterized as: little curved or direct rod shape, the long 2 μ m of typical cell, wide 0.5 μ m.The bacterium colony grey is to green on the MBA flat board, and circle is swelled, and is moistening glossy.The nematicide mechanism reason of G6 bacterial strain is decomposed the nematode body wall for forming crude protein earlier, and in body wall infection thread polypide, nematode is all decomposed the most at last.In whole nematicide process, be accompanied by protease, chitinase the effect that produces to nematode.
Stenotrophomonas maltophilia G6 is the inventor in the food nematode fungus research of carrying out, and separates obtaining from a large amount of samples, finds the insecticidal activity that it is high to the nematode tool.With the bacterial strain purifying and after carrying out multiple batches of eelworm-killing activity test, entrust institute of microbiology of Yunnan University that bacterial strain is identified, be defined as having a liking for the few food of Fructus Hordei Germinatus (narrow food) monad stenotrophomonas maltophilia G6.
The present invention is achieved in that
Enlarged culture G6 bacterial strain extracts nematicide active ingredient, carries out the test of nematicide drug effect, determines its effect to nematode.
G6 cultural method (below be weight percentage):
1, the test tube kind is cultivated
Culture medium prescription is: 1-3% yeast extract, 0.5-2% peptone, 1-4% glucose, 1.8-2% agar, remainder are water, pH7.The G6 mycelium is inoculated on the medium, cultivated 2 days down, obtain the test tube kind for 30 ℃.
2, fluid enlargement culture
The liquid culture based formulas is: 1-3% dusty yeast, 10-20% soybean-cake flour, 0.5-2% fish meal, 1-4 glucose, remainder are water, pH7.The test tube kind is inoculated in the 250ml triangular flask liquid nutrient medium, and every bottled 100ml cultivated incubation time 2 days in 30 ℃ of following shaking tables, and rotating speed is 220rpm.
3, extract the eelworm-killing activity composition
With fermentation culture medium centrifugal 20 minutes in 8000rpm/min, get supernatant, add ammonium sulfate in 1: 0.85 ratio, left standstill 2 hours in 4 ℃, 8000rpm/min is centrifugal 30 minutes then, abandons supernatant.Tris-HCl buffer solution (PH8.0) with 10mM dissolves again, to dissolve the solution that the obtains 21mm bag filter (MW:8 that packs into again, 000-14400KDa) in 20 times 10mMtris-HCl buffer solution (PH8.0), dialyse 4 times, each 3 hours, promptly get the nematicide soup.Be stored in 4 ℃ of refrigerators soup stand-by.
The test of G6 eelworm-killing activity:
1, preparation test with medicament
Cultivate the G6 bacterial strain by aforementioned liquids enlarged culture 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, but do not insert the G6 bacterial strain in the medium.
Contrast 2: in contrast with clear water.
Contrast 3: in order to prove the nematicide effective ingredient is albumen, and the reagent agent of preparation is boiled after the deactivation in contrast.
2, nematode is used in the preparation test
(1) preparation Panagrellus redivivus nematode
The P.redivivus nematode is inoculated on the medium oatmeal, cultivated 6 days down, freeze in 4 ℃ of refrigerators standby 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 sterile 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 sterile water with the nematode dilution.
(2) preparation pine wood nematode (Bursaphelenchus xylophilus)
Put into 15g through 2 days iblet of water logging bubble in the 100ml triangular flask, add water 10ml, autoclaving inserts Botrytis cinerea (Botrytis cinerea).Cultivated 5 days for 25 ℃.After treating that mycelia is paved with triangular flask, inoculation was cultivated 15 to 20 days for 28 ℃ through the pine wood nematode of 0.25% clorox surface sterilization.With sterile water nematode is washed, making content is the nematode suspension of 15/μ l.
3, test method:
(1) test of pesticide effectiveness method
Get test with medicament 200 μ l in the 1.5ml centrifuge tube, add 150 of living nematodes, centrifuge tube keeps flat, and places under 25 ℃, and Panagrellus redivivus nematode was respectively at 12 hours, 24 hours, 48 hours; Pine wood nematode is respectively at the lethality of checking the calculating nematode in 24 hours, 48 hours, 60 hours.And under light microscope, observe observation line polypide wall situation of change
Identify that dead method is: add 1-5 and drip 5%NaCl solution in handling centrifuge tube, observe after 2 minutes, dead worm is stiff, and the worm that lives is then curled or twisting.
Respectively with 3 kinds of contrast medicaments, every processing triplicate.
4, result of the test
Table 1 G6 bacterial strain is to Panagrellus redivivus action effect
Table 2 pair pine wood nematode action effect
The result shows, the test with medicament of extracting from G6 strain liquid tunning is to Panagrellus redivivus nematode and the equal tool of pine wood nematode insecticidal effect preferably, and the average mortality of nematode is more than 95%.From handling the variation of nematode, experienced from part body wall dissolving (Fig. 2) to polypide all processes (Fig. 3) of dissolving, illustrate that active ingredient is mainly the enzyme that decomposes nematode.And will be with batch test with medicament of extracting through boiling, 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 G6 is mainly enzyme to the active ingredient of nematode.
It is enzyme that G6 bacterial strain of the present invention mainly acts on composition, to nematode particularly pine wood nematode have significant toxic action.The G6 bacterial strain is grown under condition of culture of the present invention and is exceedingly fast simultaneously, has possessed good application and development prospect.
Description of drawings:
12 hours situations that are fixed of Fig. 1 display process nematode.
24 hours decomposition situations of Fig. 2 display process nematode.
36 hours decomposition situations of Fig. 3 display process nematode.
Fig. 4 shows the contrast nematode that polypide remains intact.
Embodiment:
Below be embodiments of the invention, but content of the present invention is not limited thereto.
Embodiment one:
Stenotrophomonas maltophilia G6 thalline is inoculated on the test tube agar medium inclined-plane, and culture medium prescription is: 2% yeast extract, 1.5% peptone, 2% glucose, 1.8% agar, remainder are water, and pH is 7.The G6 mycelium is inoculated on the medium, cultivated 2 days down, obtain the test tube kind for 30 ℃.
The test tube kind is inoculated into (every bottled 100ml) in the 250ml triangular flask liquid nutrient medium, and the liquid culture based formulas is: 2% dusty yeast, 15% soybean-cake flour, 1% fish meal, 3% glucose, remainder are water, pH7.Cultivated incubation time 2 days in 30 ℃ of following shaking tables, rotating speed is 220rpm.
With fermentation culture medium centrifugal 20 minutes in 8000rpm/min, get supernatant, add ammonium sulfate in 1: 0.85 ratio, left standstill 2 hours in 4 ℃, 8000rpm/min is centrifugal 30 minutes then, abandons supernatant.Tris-hcl (pH 8.0) with 10mM dissolves again, will dissolve again the solution that obtains pack into the 21mm bag filter (MW:8,000-14400KDa) in 20 times 10mMtris-hcl (pH 8.0) dialysis 4 times, promptly get the nematicide soup.
Embodiment two:
Substantially with embodiment one, difference is the liquid culture based formulas, and its prescription is: 3% dusty yeast, 10% soybean-cake flour, 2% fish meal, 1% glucose, remainder are water.
Embodiment three:
Substantially with embodiment one, difference is the liquid culture based formulas, and its prescription is: 1% dusty yeast, 20% soybean-cake flour, 0.5% fish meal, 4% glucose, remainder are water.
Claims (2)
1. the microbial inoculum with nematocide function obtains through the cultivation of test tube kind, fluid enlargement culture and after extracting eelworm-killing activity composition operation by producing bacterial strain, it is characterized in that this microbial inoculum is obtained by following steps:
A. producing bacterial strain is stenotrophomonas maltophilia (stenotrophomonas maltophilia) G6, CGMCC No:1522;
B. the test tube kind is cultivated: the G6 mycelium is inoculated into prescription is water for 1-3% yeast extract, 0.5-2% peptone, 1-4% glucose, 1.8-2% agar, remainder, on the medium of pH7, cultivated 2 days down, obtain the test tube kind for 30 ℃;
C. liquid culture: the test tube kind is inoculated in the 250ml triangular flask liquid nutrient medium, and every bottled 100ml cultivated incubation times 2 days in 30 ℃ of following shaking tables, and rotating speed is 220rpm; The liquid culture basigamy is: 1-3% dusty yeast, 10-20% soybean-cake flour, 0.5-2% fish meal, 1-4% glucose, remainder are water, pH7;
D. extract the eelworm-killing activity composition: with fermentation culture medium centrifugal 20 minutes in 8000rpm/min, get supernatant, add ammonium sulfate in 1: 0.85 ratio, left standstill 2 hours in 4 ℃, 8000rpm/min is centrifugal 30 minutes then, abandons supernatant, tris-HCl buffer solution with 10mM dissolves again, again the dissolving solution that the obtains 21mm bag filter of packing into is dialysed 4 times in 20 times 10mMtrls-HCl buffer solution, each 3 hours, promptly get the nematicide soup.
2. the application of the described microbial inoculum of claim 1 is characterized in that this microbial inoculum has toxic action to Panagrellus redivivus nematode and pine wood nematode, is applied to the biological control of plant nematode.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100106834A CN100401899C (en) | 2006-02-14 | 2006-02-14 | Bacterial agent capable of killing nematode and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100106834A CN100401899C (en) | 2006-02-14 | 2006-02-14 | Bacterial agent capable of killing nematode and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1806554A CN1806554A (en) | 2006-07-26 |
| CN100401899C true CN100401899C (en) | 2008-07-16 |
Family
ID=36838781
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2006100106834A Expired - Fee Related CN100401899C (en) | 2006-02-14 | 2006-02-14 | Bacterial agent capable of killing nematode and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100401899C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101781628B (en) * | 2009-11-11 | 2012-09-05 | 广西大学 | Nitrogen fixation stenotrophomonas maltophilia C4Y41 strain and application thereof |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102864133B (en) * | 2012-08-25 | 2015-11-25 | 安徽农业大学 | Addicted to wheat oligotrophy food sporangium proteolytic enzyme, its fermentation preparation and fermention medium |
| CN103224887B (en) * | 2013-05-07 | 2014-08-27 | 山东省环境保护科学研究设计院 | Preparation method of microbial nutrition agent for wastewater biochemical treatment |
| CN104277993A (en) * | 2014-03-12 | 2015-01-14 | 云南大学 | Actinomyces bacterial strain and application thereof |
| CN106148221B (en) * | 2016-01-19 | 2020-01-17 | 沈阳农业大学 | Stenotrophomonas bacteria for inducing peanuts to resist root-knot nematode disease |
| CN110982715B (en) * | 2019-08-05 | 2022-11-01 | 云南大学 | High-spore-yield purple-spore-bacterium-gene engineering bacterium delta PlflbD and construction method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1663394A (en) * | 2005-03-16 | 2005-09-07 | 云南大学 | Wireworm-killing biologic bacterial agent, its preparation and application |
| CN1670178A (en) * | 2005-03-16 | 2005-09-21 | 云南大学 | Monacrosporium elegans bactericidal agent with nematode killing function and its preparation method and use thereof |
| CN1673349A (en) * | 2005-03-07 | 2005-09-28 | 福建农林大学 | Lilacinus pseudo-blue mold new strain and process for preparing nematicide with shrimp shell thereof |
| CN1683514A (en) * | 2005-03-16 | 2005-10-19 | 云南大学 | Cystic monacrosporium janus prepn with nematocide function and its preparing method and use |
-
2006
- 2006-02-14 CN CNB2006100106834A patent/CN100401899C/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1673349A (en) * | 2005-03-07 | 2005-09-28 | 福建农林大学 | Lilacinus pseudo-blue mold new strain and process for preparing nematicide with shrimp shell thereof |
| CN1663394A (en) * | 2005-03-16 | 2005-09-07 | 云南大学 | Wireworm-killing biologic bacterial agent, its preparation and application |
| CN1670178A (en) * | 2005-03-16 | 2005-09-21 | 云南大学 | Monacrosporium elegans bactericidal agent with nematode killing function and its preparation method and use thereof |
| CN1683514A (en) * | 2005-03-16 | 2005-10-19 | 云南大学 | Cystic monacrosporium janus prepn with nematocide function and its preparing method and use |
Non-Patent Citations (6)
| Title |
|---|
| 一种新的杀线虫担子菌. 李国红等.云南大学学报（自然科学版），第23卷第2期. 2001 |
| 一种新的杀线虫担子菌. 李国红等.云南大学学报（自然科学版），第23卷第2期. 2001 * |
| 几种微生物杀线虫剂对烟草根结线虫病的防效试验. 祝明亮等.山地农业生物学报，第20卷第1期. 2001 |
| 几种微生物杀线虫剂对烟草根结线虫病的防效试验. 祝明亮等.山地农业生物学报，第20卷第1期. 2001 * |
| 松材线虫生物防治研究进展. 董锦艳等.植物保护，第31卷第5期. 2005 |
| 松材线虫生物防治研究进展. 董锦艳等.植物保护，第31卷第5期. 2005 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101781628B (en) * | 2009-11-11 | 2012-09-05 | 广西大学 | Nitrogen fixation stenotrophomonas maltophilia C4Y41 strain and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1806554A (en) | 2006-07-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103451128B (en) | Bacillus amyloliquefaciens and application thereof in preventing and controlling peach bleeding disease | |
| CN102952768B (en) | Bacillus, bacterial agent, preparation method and applications thereof | |
| CN100401899C (en) | Bacterial agent capable of killing nematode and application thereof | |
| Reeder et al. | Presence of banana bacterial wilt (Xanthomonas campestris pv. musacearum) in Rwanda | |
| CN101899408B (en) | Bacillus pumilus and application thereof for killing bursaphelenchus xylophilus with poison | |
| CN110066739A (en) | A kind of muscardine SB010 and its application in the common setose thistle Malaysia and China of prevention and treatment | |
| CN107881129A (en) | One bacillus amyloliquefaciens and its microbial inoculum, bacterial preparation process and application | |
| CN109609402A (en) | Te Jila bacillus Bacillus tequilensis XG18 and application | |
| CN105695364A (en) | New burkholderia cenocepacia and application thereof | |
| CN1288239C (en) | Nematode-eating fungus with nematode-killing function, preparing method and use thereof | |
| CN100401900C (en) | Wireworm-killing biologic bacterial agent, its preparation and application | |
| CN107090419A (en) | Bacillus amyloliquefaciens and its application | |
| CN102703342A (en) | Bacillus velezensis ZJ20 strain and liquid preparations thereof | |
| CN104560723B (en) | One plant thick wall bacterium spore Pu Keniya bacterial strains and its screening and application | |
| CN104263664B (en) | Candida with nematocidal activity as well as preparation method and application of candida | |
| CN102676393B (en) | Saccharopolyspora spinosa for producing spinosad and culture and application of saccharopolyspora spinosa | |
| CN1194085C (en) | Bacillus pumilus for killing nematoda and its preparation and use | |
| CN104560740B (en) | Metarhizium anisopliae and its application of one plant of preventing and treating oil tea as larva | |
| CN106399187A (en) | Marine bacteria BA-3 (Bacillus amyloliquefaciens-3) and application thereof in orchid disease prevention and treatment | |
| CN1320105C (en) | Bacillus for killing plant parasitic nematode and its preparation process and use thereof | |
| CN107118978B (en) | Endophytic fungi strain | |
| CN1317383C (en) | Cystic monacrosporium janus prepn with nematocide function and its preparing method and use | |
| CN1279160C (en) | Monacrosporium elegans bactericidal agent with nematode killing function and its preparation method and use thereof | |
| CN104293710B (en) | Streptomycete strains and combined application thereof in prevention and treatment of pepper seedling blight | |
| CN100436575C (en) | Heteromorphic Dactlella bacterial agent capable of killing nematode and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20080716 |