CN1286025A - Duokangling as antibiotic-type agricultural chemical and its preparing process - Google Patents
Duokangling as antibiotic-type agricultural chemical and its preparing process Download PDFInfo
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- CN1286025A CN1286025A CN 00119610 CN00119610A CN1286025A CN 1286025 A CN1286025 A CN 1286025A CN 00119610 CN00119610 CN 00119610 CN 00119610 A CN00119610 A CN 00119610A CN 1286025 A CN1286025 A CN 1286025A
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Abstract
An antibiotic type agricultural chemical "Duokangling" for rice, wheat or barley, cotton, rape, etc is prepared through fermentative culture to obtain the Hangzhou subspecies of Streptormyces, extracting the new antibiotic "Duokangling", and preparing agricultural chemical. Its advantages are broad spectrum, high effect, low residue, and no environmental pollution.
Description
The invention relates to the antibiotics biopesticide and intend agriculture and garden that new polyoxy pyrimidine nucleoside (claiming the biopesticide Duokangling again) and its preparation method and a kind of effective ingredient are Duokangling and grain, cotton, oily crop sterilizer, belong to the biopesticide preparing technical field.
Crop such as agriculture and garden gourd, fruit and vegetable, and grain in the past, cotton, oily crop are to use heavy metal compound preparations such as copper, mercury, arsenic, manganese, molybdenum and organochlorine, organic phosphor medicament always.But, these medicaments all are harmful to animal and human's body, soil is had contamination, and the harmful substance that remains in occurring in nature long term is in people, animal, plant and surrounding environment again, the pollution that is caused is the great social concern paid close attention to of various countries, the world today widely.Thereby caused having at present many chemical pesticides to be forbidden or limited use.Other has some countries to completely forbid and limited the application of chemical pesticide.
Forbidding and limit use with some chemical pesticides, and the appearance of some chemical pesticide pesticide resistance problems, cause the disease on the present various crops, some main diseases insect pests of the especially various diseases on gourd, fruit and vegetable, and paddy rice, barley and wheat have the problem that obviously increases the weight of.Therefore, some wide spectrums, efficient are developed in the very urgent expectation of people, and have the novel biopesticide of tight security.
In view of the above problems, order of the present invention is to provide a kind of to the effective antibiotics biopesticide of each plant species multiple diseases---Duokangling and preparation method thereof.
A kind of Duokangling as antibiotic-type agricultural chemical of the present invention and preparation method thereof is to cultivate by fermentation culture method, belongs to the production bacterium of streptomycete (Streptomyces) Hangzhou new subspecies, extracts antibiotics again from these cultures---Duokangling.The generation bacterium of this antibiotic---Duokangling is streptomyces hygroscopicus Hangzhou new subspecies (Streptomyces, abygroscopicus sibsp hangzhouensis sang eial) not.It has spectral signature and physicochemical property.The effective ingredient of therefore, the biological pesticide containing antibiotic that adopts preparation method of the present invention to produce---Duokangling is for intending polyoxy pyridimine nucleosides active substance.Adopting this biological pesticide containing antibiotic---Duokangling carries out field trial, to melon and fruit, vegetables, and grain, cotton, the damping off of oily crop, powdery mildew, anthracnose, fusarium wilt, stalk break, downy mildew, banded sclerotial blight, rice blast have significant control and fungistatic effect, and there is not any poisoning, the personnel of using there is not influence yet, to people and animal, environmentally safe harmless.
Below the present invention is described in detail:
Antibiotics biopesticide of the present invention---the employed microorganism fungus kind of Duokangling and preparation method thereof isolated strain actinomycetes from Zhejiang Hangzhou soil are numbered 2188.This bacterial classification belongs to the bacterial classification of streptomyces, or contains the bacterial classification of above-mentioned genetic material by nature and aritifical variant's bacterial strain, and its sorting technique is:
The bacterium classification method
1. morphologic observation strain culturing is cultivated 10 days with light microscope and electron microscope observation for 28 ℃ on medium such as Gao Shi starch agar, glucose asparagine agar, sucrose Czapek's agar.
2. the cultural characteristic bacterial strain is at starch---cultivated 7 days on the minimal medium, make spore suspension with sterile water, getting a ring then is inoculated on 11 kinds of medium slant, cultivate in 28 ℃ ± 1 ℃ insulating box, at 3,7,14,21 days difference of inoculation observed and recorded cultural characteristic, make the color description standard with two kinds of chromatograms.
3. physiological characteristic and biochemical analysis basic physiological test, utilization of carbon source, cell wall amino acid and sugared analysis of components are studied according to classification of actinomycetes basis and the described method of streptomycete identification handbook.
The bacterium classification result:
1. form and cultural characteristic substrate mycelium, it is dendritic to be branch, and the fibrillae of spores list is rubbed branch with the hands on synthetic medium, and from the beginning of straight or ripple is bent, ripe back forms the spiral of the different number of turns, and is spacious to tight by pine.Spore Jinyuan Garden shape is to oval.0.45～6 * 0.9～1.0 microns.Spore surface has thin short thorn.
On synthetic the cultivation aerial hyphae shallow taupe gray the sorus powdery is to the suede powdery to taupe gray, substrate mycelium is light yellow, produces the straw Huang---the palm-leaf fun yellow can dense property pigment, on potato culture, produce the soluble pigment (seeing Table 1) of shallow dish brown.
2. physio-biochemical characteristics experiments shows that 2188 bacterial strain liquefy gelatins, milk peptonize and do not solidify, the starch hydrolysis, not the subangle cellulose, do not produce hydrogen sulphide, melanin negative reaction (seeing Table 2).
3. utilization of carbon source 2188 bacterial strains aspect utilization of carbon source can utilize glucose, fructose, starch, mannose, inositol, galactose, cellobiose and aesculin.Arabinose, sucrose are utilized suspicious, do not utilize wood sugar, rhamnose, lactose, raffinose, sorbose and sodium potassium tartrate tetrahydrate (seeing table 3 for details).
4. cell wall components analysis
(1) amino acid analysis compares with 2.6 diaminopimelic acids and glycine, and 2188 strain cell wall hydrolysates are carried out full cell amino acid analysis, and this strain cell wall contains as a result: L-DAP and glycine show that this bacterial strain belongs to cell wall I type.
(2) sugared analysis of components 2188 strain whole-cell hydrolysates are analyzed its sugared composition with chromatography and are contained glucose to find no principal character sugar be galactose, arabinose, Madura sugar and wood sugar.Belong to sugared C type.
5. the antimicrobial spectrum of bacterial strain 2188 active substances
The active substance that very strong inhibition fungi is arranged in bacterial strain 2188 zymotic fluids, but a little less than the effect to bacterium.
Bacterial strain 2188 belongs to streptomyces through identifying, compares with known bacterial classification, it seems to streptomyces hygroscopicus (Streptomyces ahygroscopicus) is more not similar.
But significant difference is arranged again between them, and streptomyces hygroscopicus does not utilize aesculin, and 2188 bacterium utilize aesculin, utilization of carbon source has difference, the streptomyces hygroscopicus spore surface is smoothless in addition, and the spore surface of 2188 bacterial strains has thin short thorn, and other biochemical reactions also have difference.Therefore it is named and be streptomyces hygroscopicus Hangzhou new subspecies (Streptomyces ahygroscopicus subsp.Hangzhouensis Sang et al) not.
Duokangling antibiotics fermentation culture method:
Antibiotics biopesticide of the present invention---Duokangling can be adopted conventional fermentation equipment production, can also adopt computer program controlled operating system to carry out fermenting and producing.Cultured method both can be liquid culture, also can be solid fermentation and cultivated.In order to help suitability for industrialized production, can adopt liquid culture usually, promptly adopt the spore suspension or the liquid culture of producing bacterium to be inoculated on the medium, and produce by air agitation.
The nutrient source of the medium that fermenting and producing is used can be adopted carbon source commonly used, ammonia source and other nutrient source.Wherein carbon source can be starch, dextrin, maltose, glycerine, glucose, sucrose, inositol, galactose, mannose, aesculin etc., nitrogenous source can be peptone, soybean meal, cake powder, meat extract, yeast, rice bran, wheat skin, urea, corn steep liquor, ammonium salt, nitrate etc., and the nitrogen-containing compound of other organic-inorganics.Suitably add some mineral salt, phosphate, calcium, manganese, zinc, iron, cobalt etc., can also add animal and vegetable oil when fermentation, the mineral wet goods is made defoamer.
The cultivation temperature of producing bacterium, time, fermentation period etc. be there is no strict restriction, and the bacterial classification normal growth that uses from be applicable to production is as the criterion, and therefrom selects the highest condition of output for well.For example, the pH of medium can be 4～9, wherein near neutral better, and between 25 ℃～35 ℃ of the cultivation temperature, throughput 1: 0.5～1: 1, mixing speeds etc. are decided according to employed bacterial classification, for reaching best ferment effect.To the bacterial classification of its some mutagenic and breedings, its fermentation condition can be done suitable adjustment, and purpose is for reaching best ferment effect.
Active matter separates purification, the physicochemical characteristics of antibiotics and spectral signature in the antibiotics Duokangling tunning.
The separation of biopesticide Duokangling main active substances
1. normal phase chromatography separates
Silicic acid is adorned post with isopropyl alcohol, sample on the wet method, and isopropyl alcohol: moisture level gradient elution, Ultraviolet Detector 254nm detect, fraction collection, the detection of active composition, and detect the part that contains Duokangling with silica gel plate, and merge, obtain Duokangling (being called for short DKC-1) crude product.
2. thin-layer chromatography detection method
Thin-layer chromatography, with isopropyl alcohol: water is solvent, and the TLC plate is a parting material, ultraviolet 254nm detects.
The purifying of DKL-1
1. positive prepares thin-layer chromatography
The DKL-1 crude product, with isopropyl alcohol: it is solvent that water adds small amount of alkali, spreads silica gel plate (16 gram silica gel, 10～40um certainly, 40 milliliters of 0.5%CMC-Na distilled waters, shop 20 * 20cm, 3～4 drying at room temperature 24 hours), room temperature is launched (4 hours), scrape and get DKL-1 partly, the isopropyl alcohol wash-out concentrates, and obtains coarse-grain, after the drying, standby.
2.DKL-1 method for crystallising
Coarse-grain washs with ether, after doing, adds the isopropyl alcohol dissolving of small amount of thermal, splashes into ethyl acetate, places room temperature, slowly separates out white crystals, takes out mother liquor, volatilizes and promptly obtains DKL-1 decolouring crystallization.
3. reversed phase chromatography
The DKL-1 crystal that decoloured, with the dissolving of minute quantity distilled water, be injected into the reversed-phase preparative chromatography post, with 2～5% methanol aqueous solution wash-outs, ultraviolet 280nm detects, fraction collection, high performance liquid chromatogram detects, and flings to methyl alcohol, merge DKL-1 purity in the part more than 99%, cold doing obtained the pure product of DKL-1, for spectrometry.
The separation process figure of DKL-1
The main active constituent of Duokangling, the discriminating of physicochemical characteristics, spectral signature analysis and structure
1.DKL-1 have the uv absorption (220nm～320nm) of typical nucleoside antibiotic
1, its uv absorption is λ
Max(in MeoH) nm is 275.4 and 203nm, according to
1The HNMR spectrum, DKL-1 locates the low field below 8.0ppm, does not have the signal of proton, hangs down a place below 6.0ppm, and the signal of 2 protons is arranged.Therefore can judge that DKL-1 is not a typical purine class nucleosides and belong to the miazines nucleosides.
2. reach from the 1HNMR spectrum
1H-
1HcosY system sees that DKL-1 has an anomeric proton at 5.7ppm place, for base becomes the proton of glucoside with sugar, is judged as the α configuration from coupling constant J=3.20, and there are 6 typically sugared protons that encircle at 3.5oppm～4.0ppm place, by
1H-
1HcosY interconnects as can be seen, be hexose, from chemical shift and coupling constant and known hexose comparison, its similar D-glucose, in addition the signal of three protons of an integration being arranged at the 2.49ppm place is methyl signals, so also has a N-CH3 or CH3-C=0 part in the structure of DKL-1.
3. from DKL-1's
1The HNMR spectrum is judged can affirm its structure, and from present ESI-MS, the molecular weight of DKL-1 also has difference with Polyoxin, but its ultraviolet spectra, infrared spectrum and Polyoxin are similar, therefore may similar chromophore (base) be arranged with Polyoxin.
Sum up:
1.DKL-1 separation process set up, the purity of DKL-1 can reach more than 99%, its stability test shows DKL-1 to light, and heat is all very stable, crystallizable, be acicular crystal or white sprills, soluble in water, be dissolved in ethanol, methyl alcohol, acetone etc. on a small quantity, be insoluble to benzene, ethyl acetate, benzinum, organic solvents such as n-hexane.Acid is stabilized in pH2～3 time, can preserves 2～3 years,, be easy to carry out spectrometry the alkali instability.
2. from existing spectroscopic data ESI-MS, IRUV,
1The HNMR spectrum, and
1H-
1HCOSY, resolve to: the molecular weight of DKL-1 is 300, molecular formula C
12O
7K
2H
10, chemical constitution is: newly intend polyoxy pyridimine nucleosides material.
The biopesticide Duokangling is suitable for production technology and formulation that this product is used
The present invention as gourd, fruit and vegetable gardening class crop with grain, cotton, when oil is made antibacterial agent, can use equally known agricultural chemicals in this technical field general solid phase carrier, preparations such as liquid phase carrier and emulsifying dispersant.And can be mixed with liquid dosage form, and granule, pulvis, emulsifier, wetting powder, tablet, finish, spray etc. are formulation arbitrarily.Solid phase carrier can be the soil of clay, kaolin, bentonite, acidity in these carriers, diatomite, calcium carbonate, nitrocellulose, starch, gum Arabic etc., liquid phase carrier can be a water, hydrous ethanol, methyl alcohol, acetone, isopropyl alcohol etc. also can be methylformamide, ethylene glycol, dimethylbenzene etc.Simultaneously, in preparation, generally can suitably add some auxiliary agents, as sulfuric acid ester, the polyethylene glycol oxide of higher alcohol, alkyl allyl ether, the alkyl pi-allyl, glycol ether, the anhydrous sorbitol lauric acid of alkyl pi-allyl fat, alkylallyl sulfonate, quaternary amine, polyalkylene oxide etc. are common, the proportioning of effective ingredient in the various preparations is 1～90% in emulsion and wetting powder, is 1～20% in liquid dosage form.Be 0.1～10% in the finish, also can be suitably increased and decreased according to different application targets.
In addition, all right and other bactericide of preparation of the present invention, weed killer herbicide, insecticide, the fertilizer material, soil conditioners etc. are used.
Below, make specific description with regard to the example of manufacturing of the present invention, enforcement, test, but this is not to mean the present invention is limited to some extent.
Production Example:
Glucose 1%, starch 7%, dried yeast 1.5%, soybean meal 2%, salt 0.15%, potassium dihydrogen phosphate 0.05%, MgSO
4It is 60～70% to be made into the medium of 25 tons of fermentation tanks that the proportioning of (magnesium sulfate) 0.005% is mixed with loading amount.The biopesticide Duokangling is produced bacterial classification, be inoculated into through the conventional method sterilization and be cooled in the fermentation tank culture medium of proper temperature, ferment through air agitation in more than 60 hours.Fermented product is got its filtrate by plate-frame filtering, concentrates through vacuum film and can make 1%～20% liquid dosage form, can also extract crystallization by above-mentioned process for purification, and pulvis is made emulsion, finish etc.; Can also make pulvis, wetting powder, granule, tablet etc. by solid phase carrier.
Embodiment 1:
Liquid dosage form
With the tunning of biopesticide Duokangling, remove slag through plate compression, zymotic fluid concentrates with the vacuum film inspissator.
Detect to control respectively by fermentation titer and make 1%, 2%, 3%, 5% to 20% fluid product.
Embodiment 2, pulvis and elaboration
Zymotic fluid can be made into coarse-grain by the process for purification of above-mentioned biopesticide, and content reaches 20～45% pulvis respectively, until the elaboration that can be refined into 98%.
Embodiment 3, wetting powder
With coarse-grain and 6 parts of dodecyl sodium sulfates of 10 parts of biopesticide Duokanglings, 2 parts of diphenyl-methane one sodium disulfonate-yubans and 82 parts of potter's clay mix, and pulverizing can obtain 100 parts of preparations.
Embodiment 4, granule
10 parts of biopesticide coarse-grain knots are added 16 parts of diatomite, and 72 parts of powdered bentonites and 2 parts of dodecyl sulphates mix, and granulation promptly gets 100 parts of preparations.
Control test to powdery mildew of cucumber (or cucurbit powdery mildew)
Biopesticide Duokangling liquid dosage form of making by embodiment 1 1% or 3% Duokangling aqua, be diluted to respectively fixed concentration, evenly be sprayed onto morbidity then maybe with the plant leaves and stems of morbidity.Can also be with testing after natural powdery mildew spore and the infection-induced powdery mildew, control group, the experimental group of different spraying medicine concentrations are established in test.
Before test, carry out the inspection of disease index earlier, after carrying out dispenser again after one week of dispenser, carry out the inspection of control efficiency then, the powdery mildew disease index is checked, carry out with reference to the grade scale of powdery mildew of cucumber.
The disease index occurring degree
0 does not fall ill
10＜*≤5 * morbidity leaf area accounts for blade face %
2 5＜×≤20
3 20＜×≤40
4 40＜×≤60
5 ＜60×≤100
Result of the test: the average preventive effect poisoning of test pesticide concentration control efficiency
(ppm) Duokangling 100 96.0 does not have
93.0 do not have
95.3 94.7 do not have
94.4 Duokangling 70 94.3 does not have
100 do not have
84.9 92.2 do not have
89.6 Duokangling 50 94.1 does not have
89.9 91.5 do not have
90.2 do not have
90.8 not having 20% triadimefon 250 87.8 82.3 has
83.5 have
78.5 have
79.4 have
By evidence, biological pesticide Duokangling of the present invention provides a kind of new using In gourd, fruit and vegetable class gardening work and grain, cotton, oily crop sterilizer, this bactericide is at low concentration Lower, corps diseases is had high prevention effect, and the security of height is arranged, there is no any Poisoning. The annex explanation: 1. the cultural characteristic of table 1 Duokangling 2188 bacterial strains 2. the physiological and biochemical property of table 2 Duokangling 2188 bacterial strains 3. the utilization of carbon source of table 3 Duokangling 2188 bacterial strains 4. table 4 Duokangling 2188 strain fermentation product antimicrobial spectrums 5. the high pressure liquid chromatography of Fig. 1 Duokangling main active substances 6. the how anti-record of Fig. 2 IR infrared spectrum (KBr) 7. Fig. 3 Duokangling ultra-violet absorption spectrum 8. Fig. 4 Duokangling ESI-MS absorption spectrum 9. Fig. 5 Duokangling1HNMR spectrum 10. Fig. 6 resist more1H-
1The cultural characteristic of Hcoy spectral catalogue 1 bacterial strain 2188
The physio-biochemical characteristics of table 2 bacterial strain 2188
The utilization of carbon source of table 3 bacterial strain 2188
Carbon source kind utilizes the situation carbon source kind to utilize situation
D-R ± sweet mellow wine+
D-Fructose-inositol+
D-wood sugar-starch-
Glucose-lactose-
Sucrose ± cellobiose+
L-rhamnose-sorbose-
D-galactolipin+aesculin+
Gossypose-sodium potassium tartrate tetrahydrate-Annotate "-"-utilize; " ± "-utilize suspicious; "-"-do not utilize The antimicrobial spectrum of table 4 bacterial strain 2188-4-4 Kind microgram/promotion kind microgram/promotion
The weak wheat rust 60～80 of weak Pyricularia oryzae 60～80 Escherichia coli effect of the weak Rhizoctonia solani Kuhn 60 proteus effect of＜20 wilts 60～80 Powdery Mildews＜30 cucumber rhizoctonia rot bacterium＜30 sweet potato black rot bacterium, 80 cotton rhizoctonia solanis＜30 tobacco brown spot pathogens, 80 stem wilt bacterium, 60 sclerotinioses, 60～80 charcoal moulds, 50 downy mildew, 50 anthrax bacterias, 50～60 hay bacilluses effect
Claims (5)
1. an antibiotics, biopesticide---the preparation method of Duokangling is characterized in that cultivating the production bacterium that belongs to streptomycete (Streptormyces) Hangzhou new subspecies by fermentation culture method, again to extract the antibiotics Duokangling in these cultures.
2. antibiotics biopesticide according to claim 1---Duokangling preparation method, the generation bacterium that it is characterized in that described antibiotic Duokangling is streptomyces hygroscopicus Hangzhou new subspecies (Straptomyces abygroscopicus subsp.Hang zhouensis sang ei al) not.
3. an antibiotics biopesticide---Duokangling is characterized in that this antibiotics biopesticide---the effective ingredient of Duokangling is new polyoxy pyridimine nucleosides active substance, the tool spectral analysis feature intended.
4. a kind of antibiotics biopesticide according to claim 1---the preparation method of Duokangling is characterized in that described fermentation culture method is liquid culture or is the solid fermentation cultivation.
5. a kind of antibiotics biopesticide according to claim 4---the preparation method of Duokangling is characterized in that described liquid culture is to adopt the spore suspension or the liquid culture of producing bacterium to be inoculated on the medium, and produces by air agitation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101961014A (en) * | 2010-03-19 | 2011-02-02 | 中国计量学院 | Method for preparing microbial pesticide for preventing and controlling fungal diseases of vegetables |
| CN101223879B (en) * | 2007-11-19 | 2011-05-11 | 中国计量学院 | Method of preparing microorganisms pesticide for preventing vegetable fungi disease |
-
2000
- 2000-08-14 CN CN 00119610 patent/CN1286025A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101223879B (en) * | 2007-11-19 | 2011-05-11 | 中国计量学院 | Method of preparing microorganisms pesticide for preventing vegetable fungi disease |
| CN101961014A (en) * | 2010-03-19 | 2011-02-02 | 中国计量学院 | Method for preparing microbial pesticide for preventing and controlling fungal diseases of vegetables |
| CN101961014B (en) * | 2010-03-19 | 2013-04-24 | 中国计量学院 | Method for preparing microbial pesticide for preventing and controlling fungal diseases of vegetables |
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