CN105669691A - Compound with activity of killing nematodes and preparation method thereof - Google Patents
Compound with activity of killing nematodes and preparation method thereof Download PDFInfo
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- CN105669691A CN105669691A CN201410656578.2A CN201410656578A CN105669691A CN 105669691 A CN105669691 A CN 105669691A CN 201410656578 A CN201410656578 A CN 201410656578A CN 105669691 A CN105669691 A CN 105669691A
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Abstract
The present invention relates to a compound with an activity of killing nematodes and belongs to the technical field of microbial and biological pesticides. The compound is 4-oxabicyclo[3.2.2]non-1 (7), 5,8-triene. The preparation method is as follows: SMrs28 bacterium strain is subjected to conventional fermentation cultivation using a liquid culture medium, after fermentation liquid is filtered, the obtained filtrate passes through a D101 type macroporous resin, the obtained eluate is subjected to low pressure concentration, and the concentrated eluate is subjected to repeated silica gel and hydroxypropyl glucan gel chromatography separations. The compound provided by the invention shows the activity of killing the nematodes in an experiment of anti-nematode activity by using a liquid soaking method, and has no environmental pollution, is low in cost, and excellent in the effects of killing the nematodes.
Description
Technical field
The present invention relates to a kind of compound and its preparation method with malicious eelworm-killing activity, belong to microorganism and biological pesticide technical field.
Background technology
Plant nematode is the extremely important Plant diseases of a class, it is reported that plant nematode causes the agricultural losses (Abadetal.2008) up to about 1,500 hundred million dollars every year in worldwide. Ditylenchus destructor is one of nematode that agricultural harm is the most important, is mainly distributed in the temperate regions of many countries, the output of potato can be made to cause huge loss. In China, Ditylenchus destructor (DitylenchusDestructor) it is one of three big diseases restricting China's sweet potato production, general morbidity field underproduction 20%-50%, grave illness field even has no harvest without receipts. Pine wood nematode (Bursaphelenchusxylophilus) disease is one of forest disease of most danger in the world, belong to international important Quarantine Objects, it is classified as first of forest disease, be called as " fire of smokeless forest ". According to Hajimeetal (2001), pine wood nematode causes the financial loss of about 1,000,000,000 dollars every year in the whole world. This disease by since report, has now been distributed widely in Japan, the U.S., Korea S, Canada and Chinese etc. from 1934. In China, this disease has distributed in many provinces and regions and has spread and spread, and causes havoc to China's Forest Resources and ecotope.
At present to the control of nematode mainly based on chemical prevention, but due to chemistry nematocides majority be high poison, persistent pesticide, nontarget organism and environment are caused serious threat. Along with the progressively enhancing of people's Environmental awareness, the application of chemical agent is restricted gradually, and therefore people make every effort to find the natural medicament that can prevent and treat plant pathogeny line insect from nature. Microorganism is one of main source producing bioactive natural product, and finding the natural product with nematode cytotoxicity from the metabolite of microorganism becomes a research focus.
Summary of the invention
It is an object of the invention to provide a kind of compound and its preparation method with malicious eelworm-killing activity.
The present invention to pick up from the Stellera chamaejasme L. rhizosphere soil of the extraction Yingshan Mountains, Gansu genus bacillus (BacillusSp.) SMrs28 has carried out the research of active metabolism chemical constituent, therefrom it is separated to compound 4-oxabicyclo [3.2.2] ninth of the ten Heavenly Stems-1 (7) of a novel structure, 5,8-triolefin (4-oxabicyclo [3.2.2] nona-1 (7), 5,8-triene), retrieve through domestic and foreign literature, have no natural origin at present and about active report, also have no the open report that it is used to prepare mematocide application.
The production bacterial strain of the present invention be genus bacillus (BacillusSp.) SMrs28, this bacterial strain SMrs28 are deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 18th, 2011, and address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCCNo.5352.
Bacterial strain SMrs28 provided by the present invention belong to bacillus (Bacillus), its bacteria colony white, smooth moistening, there is gloss, gram-positive microorganism, cell elongated rod shape, Dan Sheng, gemma oval, end is raw, expands.
The present invention is by the SMrs28 bacterial strain conventional fermentation culture of liquid nutrient medium, fermented liquid is after filtering, filtrate is crossed D101 type macropore tree ester, after the elutriant low pressure obtained is concentrated, analyse through silica gel repeatedly (sherwood oil/acetone, chloroform/ethyl acetate) and hydroxypropyl dextrane gel (SephadexLH-20) (chloroform/methanol) layer, obtain 4-oxabicyclo [3.2.2] ninth of the ten Heavenly Stems-1 (7), 5,8-triolefin, the structural formula of this compound is:
。
Its physico-chemical property:
Outward appearance: colourless acicular crystal;
Solvability: be dissolved in chloroform, acetone, methyl alcohol, methyl-sulphoxide etc.; It is insoluble to sherwood oil and water etc.;
Core magnetic spectrum feature is in table 1, and its dissolution solvent is CD3OD, 400MHz, δ: ppm;
Molecular formula: C8H8O, molecular weight is 120; High resolution positively charged ion ESI-MS mass spectrum: measured value is 121.0645 [M+H]+, calculated value is 121.0648(C8H9O);
Uv-absorbing UV (MeOH) λmax201,224,278nm;
Infrared absorption IR (KBr)ν max3024,2955,2928,2879,1514,1365,1233,1051,867,817,731,556c m-1;
Table 1 compound1H and13CNMR dataa
a1H、13C data measures when 400MHz and 100HMz respectively, and solvent for use is deuterated methanol.
Experiment shows: the active compound of the present invention, in the nematicide of soaked with liquid method is tested, is 72 constantly little in the treatment time, compound pairDitylenchusDestructorWithBursaphelenchusxylophilusThere is inhibit activities. Therefore the compound of the present invention can as the application preparing nematicide preparation and precursor substance.
The advantage that the present invention has environmentally safe, cost is low, poisoning nematode effect is good.
Embodiment
Embodiment 1:
The separation preparation of the compounds of this invention:
1. the seed culture of bacterium SMrs28:
Culture medium prescription: peptone 10g, extractum carnis 3g, NaCl5g, agar 15g, distilled water 1000mL, pH7.2, change into flat board after sterilizing, by SMrs28 bacterial suspension inoculation to, on flat board, cultivating 5-7 days for 28 DEG C;
2. the liquid fermentation and culture of bacterium SMrs28:
Culture medium prescription: glucose 15g, yeast powder 10g, KH2PO46g、K2HPO4·3H2O4g、MgSO4·7H2O0.2g, distilled water 1000mL, pH7.2, by flat board strain inoculation to (every bottled 75ml) liquid nutrient medium in 150ml triangular flask, at 28 DEG C, shaking table is cultivated, rotating speed 180rpm, incubation time 2 days;
3. by filtering fermentation liquor, filtrate is adsorbed through D101 type macroporous resin, first carry out wash-out with distilled water, use 90% ethanol elution afterwards, stripping liquid concentrates laggard row silica gel column chromatography through low pressure, taking sherwood oil/acetone (10:1-1:1) as eluent gradient wash-out, the analysis of recycle silicon glue post (chloroform/ethyl acetate 10:1-2:1) repeated multiple times layer and SephadexLH-20(chloroform/methanol) separation, namely separation obtains compound.
Embodiment 2:
The compounds of this invention is detected to the test of pesticide effectiveness of Plant nematode by soaked with liquid method:
1. test with medicament
Compound is dissolved in 60 μ L methyl-sulphoxides, dilutes preparation 800 μ g mL respectively with sterile distilled water-1、400μg·mL-1、200μg·mL-1、100μg·mL-1With 50 μ g mL-1The test liquid of concentration, in this solution, the final concentration of methyl-sulphoxide is no more than 1%.Separately will containing 1%(v/v) aqueous solution of methyl-sulphoxide is in contrast.
2. the cultivation of nematode and preparation
By nematodeDitylenchusDestructorWithBursaphelenchusxylophilusBe inoculated in respectively cover with fusarium solani (Fusariumsolani) and botrytis cinerea (BotrytiscinereaPers), in potato dextrose agar (PDA), cultivate 5-7 days in 28 DEG C. After whole culture dish is covered with in nematode breeding, being inverted by culture dish, to adding 3-5mL sterilized water in culture dish lid, place more than 6h, nematode enters in sterilized water. Nematode dilution, according to nematode density under each visual field, is the nematode suspension of about 200/mL with sterile distilled water by mirror inspection under the microscope.
3. test method
By test liquid 5 μ L in 24 orifice plates, add 195 μ L nematode suspensions, as in 28 DEG C of constant incubators. Checked under an optical microscope respectively at 24,48,72 hours and calculate nemic death rate.
The method that qualification is dead: dead nematode is stiff or in " J " type, living nematode is curling twisting then. Being gone to after in sterilized water by stiff nematode, nematode is not made any reaction yet and is then accredited as death.
Mortality ratio=(verge of death is died and counted/total nematode number) × 100%;
Corrected mortality=test sample mortality ratio-comparison mortality ratio.
Not add the methyl-sulphoxide aqueous solution of sample as comparison, whole experiment repeats three times, and experiment three is parallel every time, averages, and calculates average mortality and corrected mortality.
4. experimental result
Compound pair described in table 1DitylenchusDestructorWithBursaphelenchusxylophilusThe poisoning result (%) of two kinds of nematodes is such as table 2.
Table 2
Result shows, the compounds of this invention pairDitylenchusDestructorWithBursaphelenchusxylophilusTwo kinds of nematodes have good insecticidal effect.
Claims (6)
1. one kind has the compound of malicious eelworm-killing activity, it is characterised in that the structural formula of this compound is:
。
2. the preparation method of compound as claimed in claim 1, it is characterized in that conventional for SMrs28 bacterial strain liquid nutrient medium fermentation culture, fermented liquid is after filtering, filtrate is crossed D101 type macropore tree ester, after the elutriant low pressure obtained is concentrated, it is separated with hydroxypropyl sephadex chromatography through silica gel repeatedly.
3. preparation method as claimed in claim 2, it is characterised in that the culture medium prescription of described SMrs28 seed culture is peptone, extractum carnis, NaCl, agar and distilled water, and its pH is 7.2.
4. preparation method as claimed in claim 2, it is characterised in that the culture medium prescription of described SMrs28 liquid fermentation and culture is glucose, yeast powder, KH2PO4、K2HPO4·3H2O、MgSO4·7H2O and distilled water, and its pH is 7.2.
5. preparation method as claimed in claim 2, it is characterised in that described silica gel column chromatography elutriant used is the mixed solution of sherwood oil and acetone and the mixed solution of chloroform and ethyl acetate.
6. preparation method as claimed in claim 2, it is characterised in that described hydroxypropyl sephadex chromatography elutriant used is the mixed solution of chloroform and methyl alcohol.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012115225A1 (en) * | 2011-02-25 | 2012-08-30 | Ｊｘ日鉱日石エネルギー株式会社 | Nematode attractant and method for nematode control |
| CN103773707A (en) * | 2012-10-26 | 2014-05-07 | 中国科学院兰州化学物理研究所 | Bacillus with nematocidal activity, and preparation method and application of bacillus |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012115225A1 (en) * | 2011-02-25 | 2012-08-30 | Ｊｘ日鉱日石エネルギー株式会社 | Nematode attractant and method for nematode control |
| CN103773707A (en) * | 2012-10-26 | 2014-05-07 | 中国科学院兰州化学物理研究所 | Bacillus with nematocidal activity, and preparation method and application of bacillus |
Non-Patent Citations (2)
| Title |
|---|
| 崔海燕，等: "瑞香狼毒根中异新狼毒素A和新狼毒素B对松材线虫和拟松材线虫的触杀活性研究", 《天然产物研究与开发》 * |
| 郭霞，等: "瑞香狼毒根提取物及不同溶剂萃取物对马铃薯腐烂茎线虫触杀活性研究", 《植物保护》 * |
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Inventor after: Qin Bo Inventor after: Zeng Liming Inventor after: Jin Hui Inventor after: Li Xiuzhuang Inventor after: Pan Le Inventor after: Yang Xiaoyan Inventor before: Qin Bo Inventor before: Zeng Liming Inventor before: Jin Hui Inventor before: Pan Le Inventor before: Yang Xiaoyan |
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