CN101787376B - Paecilomyces metabolite and application thereof - Google Patents
Paecilomyces metabolite and application thereof Download PDFInfo
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- CN101787376B CN101787376B CN2010100391098A CN201010039109A CN101787376B CN 101787376 B CN101787376 B CN 101787376B CN 2010100391098 A CN2010100391098 A CN 2010100391098A CN 201010039109 A CN201010039109 A CN 201010039109A CN 101787376 B CN101787376 B CN 101787376B
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Abstract
The invention relates to a paecilomyces metabolite and application thereof, which belong to the technical field of microbial medicine screening. The invention is realized by the following steps that: a production strain is paecilomyces sp.1788, and the preservation code is CGMCCNo. 3422; the paecilomyces sp.1788 is fermented and cultured by czapek culture medium conventional liquid, the fermentation fluid is concentrated at low pressure, column chromatography is carried out to water phase repeatedly, and the paecilomyces metabolite of the invention is prepared after elution. The paecilomyces metabolite of the invention has the function of killing bursaphelenchuh xylophilus, the lethal rate (ED50) in 24h is 299.5ppm, and the ED95 is 796.2ppm, and can be applied to bursaphelenchuh xylophilus preparations. Compared with the traditional bursaphelenchuh xylophilus chemical, the invention is characterized by high efficiency, low toxin, water solubility, easy preparation and the like.
Description
Technical field:
The present invention relates to a kind of Paecilomyces varioti meta-bolites and application thereof, belong to microbial medicine triage techniques field.
Background technology:
Some kinds in the paecilomyces play an important role in the controlling plant insect pest; Like the control of Paecilomyces lilacinus to plant nematodes such as SCN, root knot nematodes; Parasitic and the various insects that causes death of paecilomyces fumosoroseus and powder Paecilomyces varioti ability comprises Homoptera, lepidopteran, Diptera, Coleoptera, Hymenoptera etc.In many countries, these Paecilomyces variotis successfully are developed to biocontrol fungicide, in making up the ecological agriculture, play an important role.
Paecilomyces (Paecilomyces Bainier) belongs to Deuteromycotina (Deuteromycotina), hyphomycetes (Hyphomycetes), hyphomycetales (Hyphomycetales), Moniliaceae (Moniliaceae).Main morphological specificity is the mycelia multinuclear, and white, pearl, spread from the hair shape to dark white or brown.Conidiophore produces from aerial hyphae or the funiculus that spreads, but many kind lacks clear and definite sporophore.Have or do not have coremium to form, length variations is very big.Bottle metulae portion's column or sphere are expanded, and upwards spindle often surpasses the half the of length overall, many crooked main shafts that also depart from; It is complicated that bottle is obstructing the situation of giving birth to, or be the branch of colyliform as mould, or grow thickly brokenly on short and small branch, or singly be born on the aerial hyphae that spreads.The conidium unit cell, the column of pseudovum circle, ellipse, kidney shape or spindle, the minority kind is subsphaeroidal.Conidium is gathered into dry discrete chain usually, sometimes oblique arrangement or form irregular spore agglomerate.On the Cha Shi substratum, bacterium colony is white, pearl, greenish orange yellow, yellowish pink, fuzzy filbert, tawny or real green usually.
Pine wood nematode nineteen eighty-two finds in the Zhongshan Tomb, Nanjing first, later in succession in Jiangsu, Anhui, Guangdong and zhejiang and other places cause disaster, and almost destroyed the masson pine forest that extensively distributes in Hong Kong.Closely propagate the main media longicorn that leans on,, carry propagation like Monochamus alternatus (Monochamus alternatus); Mainly nursery stock, pine, pine tr and the pine goods etc. of allocation and transportation band epidemic disease (longicorn of band pine wood nematode) are propagated artificially at a distance.By the metainfective pine tree of pine wood nematode, needle tawny or sorrel, wilting are sagging, and the resin secretion stops, and on trunk, can be observed longicorn and invade the hole or the vestige of laying eggs, and the whole strain of sick tree is withered dead, the timber blue stain.The serious threat Timber stands.Because expansion rapidly, the natural softwood forest of scenic spot such as Mount Huang, Zhangjiajie has been constituted grave danger at present.Pine nematode also all has generation in states such as the U.S., Canada, Mexico, Japan, Korea S, Korea.Therefore, the biological prevention and control agent of developing to pine wood nematode seems particularly important.
Summary of the invention:
The object of the present invention is to provide a kind of Paecilomyces varioti meta-bolites, for exploitation pine wood nematode biocontrol fungicide lays the foundation.
The fungi that the present invention uses was Paecilomyces varioti 1788 (Paecilomyces sp.1788), and 1788 bacterial strains have been stored in China Microbial Culture Preservation Commission common micro-organisms center, address: China on November 5th, 2009. Beijing. and the Zhong Guan-cun; Preserving number: CGMCC No.3422.
The present invention is achieved in that Paecilomyces varioti (Paecilomyces sp.1788) through Cha Shi substratum conventional liq fermentation culture; After fermented liquid low pressure concentrated; The water warp is column chromatography repeatedly; Wash-out obtains Paecilomyces varioti meta-bolites of the present invention, and this Paecilomyces varioti meta-bolites is obtained by following steps:
A. producing bacterial strain is Paecilomyces sp.1788, preserving number: CGMCC No.3422;
B. the mycelium with 1788 bacterial strains is inoculated on the test tube PDA culture medium slant, cultivates 4-8 days down, obtains the test tube kind for 20-30 ℃;
C. the test tube kind is inoculated in the 250ml triangular flask of every bottled 50ml liquid nutrient medium, culture medium prescription is: peptone 10g, yeast extract paste 10g, glucose 20g, tap water 1000ml, pH nature; Culture temperature 20-30 ℃; Shaking table is cultivated, and rotating speed is 100-250rpm; Incubation time 5-15 days;
After the fermented liquid low pressure that d. will contain mycelia concentrates, extract the concentrated formation of the water position low pressure after extraction liquid concentrator successively with ETHYLE ACETATE and propyl carbinol; Also constantly stir in the acetone with 2 times of volumes of its slow adding; Leave standstill then, form water-soluble phase of acetone and precipitated phase, separate two phases again;
E. it is water-soluble colourless basically mutually until acetone to repeat several, the collecting precipitation phase; Precipitated phase repeated the polycaproamide post; With acetone and water gradient elution, pass through C-18 silicagel column and SephadexLH-20 gel column methanol-eluted fractions again, serve as with reference to merging component with the TLC plate; The active tracking detected institute and separated and obtain active ingredient, develops the color redness with 0.5% ethanol solution of ninhydrin.
Experiment shows: the tunning pine wood nematode poisoning of Paecilomyces varioti 1788 (Bursaphelenchuh xylophilus), 24 hours semilethal rate (ED
50) be 299.5 ppm, ED
95Be 796.2ppm.
Paecilomyces varioti meta-bolites of the present invention has the function of pine wood nematode poisoning (Bursaphelenchuhxylophilus), can kill the application of pine wood nematode preparation as preparation.
The present invention compares with the existing pine wood nematode medicine that kills, have efficient, low toxicity, soluble in water, be prone to characteristics such as preparation.
Embodiment:
The fermentation method for producing of active substance of the present invention:
1, the mycelium with 1788 bacterial strains is inoculated on the test tube culture medium slant, and culture medium prescription is the PDA substratum, cultivates 4 or 6 or 8 days down, obtains the test tube kind for 20 or 25 or 30 ℃;
2, the test tube kind is inoculated in the 250ml triangular flask in (every bottled 50ml) liquid nutrient medium, culture medium prescription is: peptone 10g, yeast extract paste 10g, glucose 20g, tap water 1000ml, pH nature.Culture temperature 20 or 25 or 30 ℃; Shaking table is cultivated, and rotating speed is 100-250 rpm; Incubation time 5 or 10 or 15 days.
After the fermented liquid low pressure that 3, will contain mycelia concentrates, extract the concentrated formation of the water position low pressure after extraction liquid concentrator successively with ETHYLE ACETATE and propyl carbinol; Also constantly stir in the acetone with 2 times of volumes of its slow adding; Leave standstill then, form water-soluble phase of acetone and precipitated phase, separate two phases again.It is water-soluble colourless basically mutually until acetone to repeat several, the collecting precipitation phase.Precipitated phase repeated the polycaproamide post; With acetone and water gradient elution, pass through C-18 silicagel column and SephadexLH-20 gel column methanol-eluted fractions again, serve as with reference to merging component with the TLC plate; The active tracking detected the active ingredient that is separated to, and develops the color red with 0.5% ethanol solution of ninhydrin.
Active ingredient of the present invention is to the test of pesticide effectiveness of pine wood nematode
1, test with medicament:
Use the sterile distilled water dilution for being suitable for concentration active ingredient.
Use the sterile distilled water dilution for being suitable for concentration nonvaccinated fermention medium as contrast.
2, nematode is used in the preparation test
(Botrytis cinerea) is connected on the PDA flat board with Botrytis cinerea; 25 ℃ are cultured to and cover with flat board, insert a substratum that has pine wood nematode (B.xylophilus), cultivate mycelia for 25 ℃ and disappear; Nematode is covered with flat board, and it is subsequent use to place 4 ℃ of refrigerators to preserve then.Wash out by the graceful funnel method of shellfish during use, regulate the nematode suspension to about 300/ml with sterilized water.
3, TP
Get 2ml specimen or control sample in the petridish of 6cm diameter, add the nematode suspension (about 100) of 0.3ml with liquid-transfering gun.When measuring activity, under anatomical lens, observe in different time (12h and 24h), record dead wire borer population (motionless with physical stimulation is standard) and total nematode number, calculation sample is to nematode " sample lethality rate " and " contrast mortality ratio ".A sample is established two repetitions, obtains " the correction lethality rate " of this bacterial strain at last.The correction lethality rate of strains tested calculates by following formula:
Sample average lethality rate (%)=∑ (the bus borer population that dead wire borer population/observation that observation count down to count down to) * 100%/n;
Proofread and correct lethality rate (%)=sample average lethality rate (%)-contrast average lethality rate (%)
Embodiment 1: Paecilomyces varioti 1788 fermented liquids are to the toxic action of pine wood nematode
Cultured liquid fermentate after with vacuum filtration clear solution, this solution is evaporated to dried active substance at 60-70 ℃, is dissolved in the sample that sterile distilled water is mixed with 5mg/ml, carries out the test of pesticide effectiveness by above-mentioned TP.Tabulation is depicted as corrected mortality.
Table 1 fermented liquid is to the toxic action of B.xylophilus nematode
The result shows that the liquid fermentation production of Paecilomyces varioti 1788 has significant toxic action to pine wood nematode, and lethality rate reaches more than 92%.
Embodiment 2: active ingredient is to the toxic action of pine wood nematode
The active ingredient that separation is obtained is dissolved in sterile distilled water, dilutes the solution for 500ppm.
Carry out the test of pesticide effectiveness by above-mentioned TP.Tabulation is depicted as corrected mortality.
Table 2 active ingredient is to the toxic action of B.xylophilus nematode
The result shows that the active ingredient that separation obtains reaches more than 80% at 24 hours the lethality rate of B.xylophilus nematode.Has tangible eelworm-killing activity.
Embodiment 3: active ingredient is to the semilethal rate ED of pine wood nematode
50
Embodiment 3 methods are with embodiment 2, and difference is: the 2.5mg active ingredient is dissolved in small amount of aseptic zero(ppm) water, is formulated as the aqueous solution of 1000ppm, semilethal rate (ED is measured in sesquialter dilution subsequently
50) and 95% lethality rate (ED
95).
The ED of table 3 pair B.xylophilus nematode
50And ED
95
The result shows that active ingredient was at 24 hours semilethal rate (ED to the B.xylophilus nematode
50) for being 299.5 ppm, 95% lethality rate (ED
95) be 796.2 ppm.
Claims (3)
1. the working method of a Paecilomyces varioti meta-bolites is characterized in that the step of this working method is following:
A. producing bacterial strain is Paecilomyces sp.1788, preserving number: CGMCC No.3422;
B. the mycelium with 1788 bacterial strains is inoculated on the test tube PDA culture medium slant, cultivates 4-8 days down, obtains the test tube kind for 20-30 ℃;
C. the test tube kind is inoculated in the 250ml triangular flask of every bottled 50ml liquid nutrient medium, culture medium prescription is: peptone 10g, yeast extract paste 10g, glucose 20g, tap water 1000ml, pH nature; Culture temperature 20-30 ℃; Shaking table is cultivated, and rotating speed is 100-250rpm; Incubation time 5-15 days;
After the fermented liquid low pressure that d. will contain mycelia concentrates, extract the concentrated formation of the water position low pressure after extraction liquid concentrator successively with ETHYLE ACETATE and propyl carbinol; Also constantly stir in the acetone with 2 times of volumes of its slow adding; Leave standstill then, form water-soluble phase of acetone and precipitated phase, separate two phases again;
E. it is water-soluble colourless basically mutually until acetone to repeat several, the collecting precipitation phase; Precipitated phase repeated the polycaproamide post; With acetone and water gradient elution, pass through C-18 silicagel column and SephadexLH-20 gel column methanol-eluted fractions again, serve as with reference to merging component with the TLC plate; The active tracking detected institute and separated and obtain active ingredient, develops the color redness with 0.5% ethanol solution of ninhydrin.
2. production bacterial strain Paecilomyces sp.1788 who produces the Paecilomyces varioti meta-bolites, preserving number: CGMCC No.3422.
3. the production bacterial strain of claim 2 has the application in the pesticide preparation of function of pine wood nematode poisoning (Bursaphelenchuh xylophilus) in preparation.
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| CN2010100391098A CN101787376B (en) | 2010-01-05 | 2010-01-05 | Paecilomyces metabolite and application thereof |
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| CN2010100391098A CN101787376B (en) | 2010-01-05 | 2010-01-05 | Paecilomyces metabolite and application thereof |
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| CN101787376A CN101787376A (en) | 2010-07-28 |
| CN101787376B true CN101787376B (en) | 2012-05-23 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1422292A1 (en) * | 2001-08-17 | 2004-05-26 | Hangzhou Tsbio Science & Technology Co., Ltd. | Fermentation product of cyptoporus volvatus and its preparation method and use |
| CN1827633A (en) * | 2004-10-30 | 2006-09-06 | 浙江省农业科学院 | Process for extracting N6-(2-ethoxyl) adenosine from Paecilomyces Cicadae Samson culture |
| CN101041809A (en) * | 2007-02-14 | 2007-09-26 | 贵州大学 | Anti-drought ultraviolet resistant long-storage cicadae penicillium notatum strain and application thereof |
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2010
- 2010-01-05 CN CN2010100391098A patent/CN101787376B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1422292A1 (en) * | 2001-08-17 | 2004-05-26 | Hangzhou Tsbio Science & Technology Co., Ltd. | Fermentation product of cyptoporus volvatus and its preparation method and use |
| CN1827633A (en) * | 2004-10-30 | 2006-09-06 | 浙江省农业科学院 | Process for extracting N6-(2-ethoxyl) adenosine from Paecilomyces Cicadae Samson culture |
| CN101041809A (en) * | 2007-02-14 | 2007-09-26 | 贵州大学 | Anti-drought ultraviolet resistant long-storage cicadae penicillium notatum strain and application thereof |
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