CN105669691B - A kind of compound with malicious eelworm-killing activity and preparation method thereof - Google Patents
A kind of compound with malicious eelworm-killing activity and preparation method thereof Download PDFInfo
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- CN105669691B CN105669691B CN201410656578.2A CN201410656578A CN105669691B CN 105669691 B CN105669691 B CN 105669691B CN 201410656578 A CN201410656578 A CN 201410656578A CN 105669691 B CN105669691 B CN 105669691B
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Abstract
The present invention relates to a kind of compounds with malicious eelworm-killing activity, belong to microorganism and biological pesticide technical field.The compound is 4 oxabicyclos [3.2.2] nonyl 1 (7), 5,8 triolefins, preparation method is by SMrs28 bacterial strain fluid nutrient medium normal fermentation cultures, zymotic fluid is after filtering, filtrate is crossed into D101 type macropore resins, after obtained eluent low pressure concentration, is detached through silica gel repeatedly and hydroxypropyl sephadex chromatography.Compound of the present invention shows the nematicide activity of poison in the anti-nematode activity test of liquid infusion method, has the advantages that environmentally safe, at low cost, poisoning nematode effect is good.
Description
Technical field
The present invention relates to a kind of compounds with malicious eelworm-killing activity and preparation method thereof, belong to microorganism and biological agriculture
Medicine technical field.
Background technology
Plant nematode is a kind of very important plant disease, it was reported that plant nematode is every year in whole world model
It causes to be up to about 150,000,000,000 dollars of agricultural losses (Abad et al. 2008) in enclosing.Ditylenchus destructor is to agriculture
Industry endangers one of most important nematode, is mainly distributed on the temperate regions of many countries, can make potato yield cause it is huge
Loss.In China, Ditylenchus destructor（Ditylenchus Destructor）It is restrict the production of China sweet potato three
One of major disease, the general field underproduction 20% -50% of falling ill, grave illness field even has no harvest without receipts.Bursaphelenchus xylophilus
（Bursaphelenchusxylophilus）Disease is one of forest disease most dangerous in the world, belongs to international important quarantine
Object is classified as first of forest disease, is referred to as " fire of smokeless forest ".According to Hajime et al (2001), pine line
Worm causes about 1,000,000,000 dollars of economic loss in the whole world every year.The disease has been distributed widely in day since 1934 are reported
Sheet, the U.S., South Korea, Canada and China etc..In China, which is distributed in more provinces and regions and spreads sprawling, gives China forest
Resource and ecological environment do great damage.
The prevention of nematode is mainly controlled by chemical pesticides at present, but since chemical nematicides majority is high poison, high residual
Pesticide is stayed, serious threat is caused to nontarget organism and environment.With the gradually enhancing of people's environmental consciousness, chemical agent is answered
With being restricted gradually, therefore people make every effort to find the natural medicament that can prevent plant pathogeny line insect from nature.Microorganism
It is one of main source for generating bioactive natural product, is found from the metabolin of microorganism natural with nematode cytotoxicity
Product becomes a research hotspot.
Invention content
The purpose of the present invention is to provide a kind of compounds with malicious eelworm-killing activity and preparation method thereof.
The present invention is to picking up from the bacillus in the extraction Yingshan Mountains Chinese Stellera Root rhizosphere soil of Gansu（Bacillussp.）
SMrs28 has carried out the research of active metabolism chemical constituent, is therefrom isolated to the compound 4- oxa-s two of a structure novel
Ring [3.2.2] nonyl- 1 (7), 5,8- triolefins（4-oxabicyclo[3.2.2]nona-1(7),5,8-triene）, through domestic foreign language
Retrieval is offered, has no natural origin and the report in relation to activity at present, also has no its public affairs that be used to prepare mematocide application
Open report.
The production bacterial strain of the present invention is bacillus（Bacillussp.）SMrs28, bacterial strain SMrs28 is in 2011
It was deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 18, address is Chaoyang District, Beijing City north
The institute 3 of occasion West Road 1, deposit number are CGMCC No. 5352.
Bacterial strain SMrs28 provided by the present invention belongs to bacillus（Bacillus）, bacteria colony white, smooth moistening,
With gloss, gram-positive bacteria, cell elongated rod shape, Dan Sheng, gemma oval, end is given birth to, is expanded.
The present invention by the fluid nutrient medium normal fermentation culture of SMrs28 bacterial strains, zymotic fluid after filtering, by filtrate mistake
D101 type macropore resins, after obtained eluent low pressure concentration, through silica gel repeatedly（Petroleum ether/acetone, chloroform/ethyl acetate）With
Hydroxypropyl sephadex（Sephadex LH-20）（Chloroform/methanol）Chromatography, obtains 4- oxabicyclos [3.2.2] nonyl- 1 (7),
5,8- triolefins, the structural formula of the compound are：
。
Its physicochemical property：
Appearance：Colourless acicular crystal；
Dissolubility：It is dissolved in chloroform, acetone, methanol, dimethyl sulfoxide etc.；Insoluble in petroleum ether and water etc.；
Nuclear-magnetism spectrum signature is shown in Table 1, dissolution solvent CD3OD, 400MHz, δ:ppm；
Molecular formula：C8H8O, molecular weight 120；High-resolution cation ESI-MS mass spectrums：Measured value is 121.0645 [M+H
]+, calculated value 121.0648（C8H9O）；
UV absorption UV (MeOH) λmax201,224,278nm；
Infrared absorption IR (KBr)ν max3024,2955,2928,2879,1514,1365,1233,1051,867,817,
731,556 cm-1；
1 compound of table1H and13C NMR datasa
a 1H、13C data measures under the conditions of 400MHz and 100HMz respectively, and solvent for use is deuterated methanol.
Experiment shows：The reactive compound of the present invention is 72 in processing time in the anti-nematode test of liquid infusion method
During hour, compound pairDitylenchus DestructorWithBursaphelenchusxylophilusThere is inhibitory activity.Cause
This compound of the present invention can be as the application for preparing nematicide preparation and precursor substance.
The present invention has the advantages that environmentally safe, at low cost, poisoning nematode effect is good.
Specific embodiment
Embodiment 1：
It is prepared by the separation of the compounds of this invention：
1. the seed culture of bacterium SMrs28：
Culture medium prescription：10 g of peptone, 35 g of g, NaCl of beef extract, 15 g of agar, distilled water 1000 mL, pH
7.2, tablet is changed into after sterilizing, by SMrs28 bacterial suspension inoculations to tablet, 28 DEG C are cultivated 5-7 days；
2. the liquid fermentation and culture of bacterium SMrs28：
Culture medium prescription：15 g of glucose, dusty yeast 10 g, KH2PO4 6 g、K2HPO4·3H2O 4 g、MgSO4·
7H20.2 g of O, 1000 mL, pH 7.2 of distilled water, tablet strain is inoculated into 150ml triangular flasks（Per bottled 75 ml）Liquid
Body culture medium, the shaking table culture at 28 DEG C, rotating speed 180rpm, incubation time 2 days；
3. by filtering fermentation liquor, filtrate is adsorbed through D101 type macroporous resins, is first eluted with distilled water,
Afterwards with 90% ethanol elution, stripping liquid carries out silica gel column chromatography after low pressure concentrates, with petroleum ether/acetone (10:1-1:1) to wash
De- agent gradient elution, then use silicagel column（Chloroform/ethyl acetate 10:1-2:1）Repeated multiple times chromatography and Sephadex LH-20（Chlorine
Imitative/methanol）Separation, i.e., isolated compound.
Embodiment 2：
The test of pesticide effectiveness of the compounds of this invention to Plant nematode is detected with liquid infusion method：
1. experiment medicament
Compound is dissolved in 60 μ L dimethyl sulfoxides, is diluted respectively with sterile distilled water and prepares 800 μ gmL-1、400 μg·
mL-1、200 μg·mL-1、100 μg·mL-1With 50 μ gmL-1The test liquid of concentration, in the solution, the end of dimethyl sulfoxide
Concentration is no more than 1%.1% will separately be contained（v/v）The aqueous solution of dimethyl sulfoxide is as control.
2. the culture and preparation of nematode
By nematodeDitylenchus DestructorWithBursaphelenchusxylophilusIt is inoculated in and covers with respectively
Fusarium solani（Fusariumsolani）And botrytis cinerea（Botrytis cinereaPers）Potato dextrose agar training
Support base（PDA）In, it is cultivated 5-7 days in 28 DEG C.After entire culture dish is covered in nematode breeding, culture dish is inverted, to culture dish
3-5 mL sterile waters are added in lid, place 6 more than h, nematode enters in sterile water.Microscopy under the microscope, according to each visual field
Nematode is diluted to the nematode suspension of about 200/mL with sterile distilled water by lower nematode density.
3. test method
By 5 μ L of test liquid in 24 orifice plates, 195 μ L nematode suspensions are added in, as in 28 DEG C of constant incubators.Respectively at
24th, it checks under an optical microscope within 48,72 hours and calculates nemic death rate.
Identify dead method：Dead nematode is stiff or in " J " type, and living nematode then crimps twisting.Stiff nematode is turned
After into sterile water, nematode does not make any reaction and is then accredited as death yet.
The death rate=（The verge of death dies number/bus borer population）×100%；
Corrected mortality=test sample the death rate-control death rate.
Using the dimethyl sulfoxide aqueous solution for not being loaded product as control, entirely test in triplicate, experiment three is parallel every time, takes
Average value calculates average mortality and corrected mortality.
4. experimental result
Compound pair described in table 1Ditylenchus DestructorWithBursaphelenchusxylophilusTwo kinds
The poisoning result of nematode（%）Such as table 2.
Table 2
The result shows that the compounds of this invention pairDitylenchus DestructorWithBursaphelenchusxylophilusTwo kinds of nematodes have preferable insecticidal effect.
Claims (6)
1. a kind of compound with malicious eelworm-killing activity, it is characterised in that the structural formula of the compound is：
。
2. the preparation method of compound as described in claim 1, it is characterised in that by SMrs28 bacterial strains fluid nutrient medium routine
Fermented and cultured, zymotic fluid cross D101 type macroreticular resins after filtering, by filtrate, after obtained eluent low pressure concentration, through repeatedly
Silica gel and the separation of hydroxypropyl sephadex chromatography.
3. preparation method as claimed in claim 2, it is characterised in that the culture medium prescription of the seed culture of the SMrs28 is
Peptone, beef extract, 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 the SMrs28 liquid fermentation and cultures
For glucose, dusty yeast, 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 eluent used in the silica gel column chromatography for petroleum ether and
The mixed liquor and chloroform of acetone and the mixed liquor of ethyl acetate.
6. preparation method as claimed in claim 2, it is characterised in that the elution used in the hydroxypropyl sephadex chromatography
Liquid is chloroform and the mixed liquor of methanol.
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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 |
-
2014
- 2014-11-18 CN CN201410656578.2A patent/CN105669691B/en active Active
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 |
|---|
| 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;崔海燕，等;《天然产物研究与开发》;20140531;第26卷(第5期);第639-644页 * |
| 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;郭霞，等;《植物保护》;20110228;第37卷(第1期);第128-131页 * |
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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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