CN101300980B - Biological pesticide containing antibiotic and preparation thereof - Google Patents
Biological pesticide containing antibiotic and preparation thereof Download PDFInfo
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- CN101300980B CN101300980B CN2008100608083A CN200810060808A CN101300980B CN 101300980 B CN101300980 B CN 101300980B CN 2008100608083 A CN2008100608083 A CN 2008100608083A CN 200810060808 A CN200810060808 A CN 200810060808A CN 101300980 B CN101300980 B CN 101300980B
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Abstract
The present invention relates to an antibiotic biological pesticide, a method for preparation and application thereof, characterized in that the active component of the biological pesticide is agricultural antibiotic HA97502 with the molecular formula of C25H37O4N and the chemical structural formula as follows. In preparation, the microorganism zymolysis technique is adopted, wherein the production bacterial is streptomyces aburayiensis HA97502. As an agricultural insecticide, the biological pesticide exhibits specific effects to important destructive insects in agricultural production, such as red spiders, aphids, cabbage worms etc.
Description
Technical field:
The present invention relates to a kind of biological pesticide containing antibiotic and preparation method thereof, belong to the microbial fermentation engineering technical field.
Background technology:
Damage by disease and insect brings about great losses to agricultural production every year, and in order to realize high and stable yields, people mainly adopt chemical pesticide to control always.Chemical pesticide has advantages such as preventive effect height, speed is fast, the insecticidal and antibacterial spectrum is wide, cost is low, use is simple, thereby has obtained developing rapidly, and becomes the important means that the agricultural production good harvest is captured in prevention and elimination of disease and pests.According to statistics, China in recent years the chemical pesticide control area account for the ratio of total control area up to about 95%.Because using for a long time, in a large number and repeatedly of chemical pesticide brought the pollution to soil, water body and atmosphere; Residue of pesticide increase in the agricultural byproducts, have also directly endangered human health; Simultaneously, the pesticide resistance of insect and germ is constantly deepened and is enlarged, and existing at least 500 various pests in the whole world have produced resistance to agricultural chemicals, and the pest resistance that has is up to several ten thousand times; In addition, chemical pesticide has has also killed and wounded natural enemy and other beneficial organism in kill pests, destroyed the ecological balance, causes that insect is rampant more.Like this, have to strengthen the amount of application and the frequency of agricultural chemicals, thus the vicious circle that causes chemical pesticide to use.China belongs to man-land relationship area at full stretch, and its outlet is to greatly develop biopesticide.
Goal of the invention:
The objective of the invention is to overcome the prior art defective, a kind of new biopesticide is provided, replace existing chemical pesticide to be used to prevent and treat crop pests such as two spotted spider mite, aphid, cabbage caterpillar.
The technical scheme of taking for achieving the above object:
Biopesticide of the present invention, its active effective ingredient are agricultural anti-HA97502, and molecular formula is C
25H
370
4N, chemical structural formula is:
Its preparation adopts A Bula streptomycete HA97502 (Streptomyces aburaviensis HA97502) for producing bacterium, produces by the microbial fermentation culture technique.
Particular content of the present invention:
1. the generation dientification of bacteria:
(1) morphological feature: bacterial strain HA97502 Gram-positive; Growth is after 7 days on medium such as synthetic No. 1 agar of Gao Shi and oat agar, and substrate mycelium physically well develops, and no tabula does not rupture, multi-branched; The aerial hyphae growth is abundant, is differentiated to form fibrillae of spores; Fibrillae of spores is until gentle song, spore ellipse, smooth surface.
(2) cultural characteristic: the cultural characteristic of bacterial strain HA97502 is seen specification subordinate list 1:
(3) cell wall type analysis: the full cell hydrolyzate of bacterial strain HA97502 contains L, L-DAP (L, L-diaminopimelic acid Diaminopimelic acid), atypism sugar; Cell wall belongs to the I type, sugared type C.
(4) physiological and biochemical property: the physiological and biochemical property of bacterial strain is seen specification subordinate list 2.
(5) 16S rDNA sequence analysis: the 16S rDNA sequence of bacterial strain HA97502 is seen sequence table:
Show that with the result relatively of correlated series Blast among the GenBank this bacterial strain belongs to streptomyces; Compare with the relevant bacterial strain of delivering at present, bacterial strain HA97502 is consistent with the 16S rDNA sequence of A Bula streptomycete (Streptomyces aburaviensis), and autoploidy is 100%.
According to morphological feature, cell-wall components and 16S rDNA sequence analysis, bacterial strain HA97502 belongs to streptomyces; Its morphological feature, 16S rDNA sequence and physiological and biochemical property are all highly similar to the A Bula streptomycete.So bacterial strain HA97502 is accredited as A Bula streptomycete (Streptomyces aburaviensis).
2. effective ingredient is identified:
(1) extraction of effective ingredient separates:
Zymotic fluid after filtration, solvent extraction, silica gel column chromatography separate and high-efficient liquid phase chromatogram purification promptly gets pure product.Idiographic flow is seen Figure of description 1.
(2) structure of effective ingredient demonstration:
The ultraviolet of the anti-HA97502 of farming has three absworption peaks, and λ max (in MeOH) is 233,238 and 267nm, with piericidin A
1Similar.The hydrogen nuclear magnetic resonance spectrum of the anti-HA97502 of farming (
1HNMR), the heterocycle of basic framework and linear fraction and piericidin A
1Unanimity, its difference mainly is the branched chain methyl part.The anti-HA97502's of farming
1The HNMR spectrum has two CH
3The chemical shift of proton absworption peak is identical, all at the 1.74ppm place, and is unimodal, and this two CH are described
3Link to each other the concrete structure See Figure with same olefinic carbon.
3. preparation technology:
(1) produce bacterial classification:
The used microorganism fungus kind of the present invention is A Bula streptomycete HA97502 (Streptomyces aburaviensisHA97502), be preserved in CGMCC (China Committee for Culture Collection of Microorganisms common micro-organisms center), preserving number CGMCCNO.2297, preservation date is on December 14th, 2007, and the preservation address is the Datun Road, Chaoyang District, Beijing City.
(2) medium:
1. seed slant medium:
KNO
30.1%, K
2HPO
40.05%, MgSO
40.05%, NaCI 0.05%, soluble starch 2.0%, agar 2.0%, peptone 0.1%, pH 7.2～7.4.
2. fermentation medium:
Nutrient source in the medium adopts carbon source and nitrogenous source commonly used.Wherein carbon source can be corn flour, starch, dextrin, sucrose, glucose, maltose etc.; Nitrogenous source can be corn steep liquor, urea, peptone, soybean cake powder, dusty yeast etc.Add some other nutritions on this basis again, as salt, phosphate, sylvite, calcium salt, magnesium salts etc.
(3) condition of culture:
Cultivation temperature is 22-34 ℃, and the cultivation pH value is 4-10, and incubation time is 30～124 hours.
(4) product post processing:
Culture fluid through centrifugal or filter thalline, thalline concentrates after with solvent extraction, or further carries out after silica gel column chromatography etc. separates, and can be mixed with the pesticidal preparations product of desired content.
Figure of description 2 is seen in concrete technological process.
Remarkable advantage of the present invention:
1. wide spectrum: the field efficacy experiment shows that agricultural anti-HA97502 has good control efficiency (more than 80%) to various crop insects such as two spotted spider mite, aphid, cabbage caterpillars.
2. efficient: Toxicity Determination shows that agricultural anti-HA97502 promptly has toxic action to the various crop insect under low concentration, and measurement result is citrus red mite LC
502.3385ppm, melon aphid LC
501.6819ppm, black peach aphid LC
502.4222ppm, cabbage caterpillar LC
505.0556ppm.
3. inexpensive: the fermentation medium main component is common agricultural byproducts, and fermentation period is also shorter, thereby production cost is lower.
Description of drawings:
Fig. 1 is pure product separation process figure of the present invention;
Fig. 2 is a product preparation process flow chart of the present invention.
Embodiment:
The present invention has following examples:
Embodiment one: the preparation of agricultural anti-HA975021% preparation
In 5 tons of fermentation tanks, be made into 3.5 tons of medium (pH7.2～7.4) by the proportioning of sweet potato starch 3%, corn flour 2%, soybean cake powder 1.5%, urea 0.5%, glucose 0.5%, dusty yeast 0.5%, calcium carbonate 0.5%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.05%.Be cooled to 28 ℃ after the sterilization according to a conventional method, inoculate agricultural anti-HA97502 and produce bacterium, promptly got the zymotic fluid of about 1500 μ g/ml that tire through 28 ℃ of aerobic culture in 58 hours.This zymotic fluid is through plate compression, and thalline is with 95% industrial alcohol lixiviate of 1.5 times of fermentating liquid volumes three times, and leaching liquor is removed alcohol through vacuum concentration, be diluted to the 10000-11500 μ g/ml that tires after, packing is 1% formulation products.
Embodiment two agricultural anti-HA97502 control eggplant two spotted spider mite
(1) reagent agent and concentration:
Reagent agent is 500 times, 650 times, 800 times dilutions of 1% agricultural anti-HA97502 preparation, and the contrast medicament is 1000 times of dilutions of 20% amitraz milk oil, and blank is a clear water.
(2) test method:
Trial crops is an eggplant, and test area is 666.7m
2, totally 4 processing repeat 3 times, and district's group is arranged at random.Adopt Shandong to produce " bodyguard " knapsack hand sprayer, consumption is by every 666.7m
2The 45Kg amount of liquid medicine is carried out the complete stool even spraying.Select 3 points at random before spraying medicine, every point selection 5 strains are as fixed point investigation strain, and every strain fixed point is investigated 5 blades in top borer population alive.24,48,72 hours remaining borer populations alive of investigation behind the spray medicine calculate go down rate and proofread and correct preventive effect of insect population.
Insect population rate (%)=[borer population of living before (borer population of living after the borer population-dispenser of living before the dispenser)/dispenser] * 100 of going down
Proofread and correct preventive effect (%)=[(the treatment region insect population rate of going down-check plot insect population go down rate)/(100-check plot insect population go down rate)] * 100
(3) result of the test:
By following table as can be seen, 500 times of dilutions of 1% agricultural anti-HA97502 preparation have good preventive effect to the eggplant two spotted spider mite, promptly have an effect in 24 hours behind the spray medicine, and preventive effect was 93.4% in 72 hours, was better than 1000 times of dilutions of 20% amitraz milk oil (90.2%).650 times, 800 times dilutions of 1% agricultural anti-HA97502 preparation are respectively 85.1%, 75.9% to 72 hours preventive effects of eggplant two spotted spider mite.
The anti-HA97502 control of farming eggplant two spotted spider mite field efficacy
Embodiment three: agricultural anti-HA97502 control cabbage caterpillar
(1) reagent agent and concentration:
Reagent agent is 500 times, 650 times, 800 times dilutions of 1% agricultural anti-HA97502 preparation, and the contrast medicament is 2500 times of dilutions of 2.5% deltamethrin milk oil, and blank is a clear water.
(2) test method:
Trial crops is a wild cabbage, and test area is 666.7m
2, totally 4 processing repeat 3 times, and district's group is arranged at random.Adopt Shandong to produce " bodyguard " knapsack hand sprayer, consumption is by every 666.7m
2The 45Kg amount of liquid medicine is carried out the complete stool even spraying.Diagonal 5 point samplings are adopted in investigation, every 2 strain, and 10 strains are investigated in every sub-district.Investigation insect population radix before the spray medicine, 1 day, 2 days, 5 days remaining borer populations alive of investigation behind the spray medicine calculate go down rate and proofread and correct preventive effect of insect population, and computational methods are with embodiment two.
(3) result of the test:
The anti-HA97502 control of farming cabbage caterpillar field efficacy
As can be seen from the above table, 500 times of liquid of 1% agricultural anti-HA97502 preparation to cabbage caterpillar after 5 days preventive effect be 85.1%, close with 2500 times of dilutions of 2.5% deltamethrin milk oil.
The cultural characteristic of subordinate list 1 bacterial strain HA97502
The physiological and biochemical property of subordinate list 2 bacterial strain HA97502
Sequence table
CTTAACACAT?GCAAGTCGAA?CGGTGAAGCC?CTTCGGGGTG?GATCAGTGGC?GAACGGGTGA 60
GTAACACGTG?GGCAATCTGC?CCTGCACTCT?GGGACAAGCC?CTGGAAACGG?GGTCTAATAC 120
CGGATATGAC?CTTCCTCCGC?ATGGGGGTTG?GTGGAAAGCT?CCGGCGGTGC?AGGATGAGCC 180
CGCGGCCTAT?CAGCTTGTTG?GTGGGGTAAT?GGCCTACCAA?GGCGACGACG?GGTAGCCGGC 240
CTGAGAGGGC?GACCGGCCAC?ACTGGGACTG?AGACACGGCC?CAGACTCCTA?CGGGAGGCAG 300
CAGTGGGGAA?TATTGCACAA?TGGGCGAAAG?CCTGATGCAG?CGACGCCGCG?TGAGGGATGA 360
CGGCCTTCGG?GTTGTAAACC?TCTTTCAGCA?GGGAAGAAGC?GCAAGTGACG?GTACCTGCAG 420
AAGAAGCACC?GGCTAACTAC?GTGCCAGCAG?CCGCGGTAAT?ACGTAGGGTG?CGAGCGTTGT 480
CCGGAATTAT?TGGGCGTAAA?GAGCTCGTAG?GCGGCCTGTC?GCGTCGGATG?TGAAAGCCCG 540
GGGCTTAACC?CCGGGTCTGC?ATTCGATACG?GGCAGGCTAG?AGTGTGGTAG?GGGAGATCGG 600
AATTCCTGGT?GTAGCGGTGA?AATGCGCAGA?TATCAGGAGG?AACACCGGTG?GCGAAGGCGG 660
ATCTCTGGGC?CATTACTGAC?GCTGAGGAGC?GAAAGCGTGG?GGAGCGAACA?GGATTAGATA 720
CCCTGGTAGT?CCACGCCGTA?AACGTTGGGA?ACTAGGTGTT?GGCGACATTC?CACGTCGTCG 780
GTGCCGCAGC?TAACGCATTA?AGTTCCCCGC?CTGGGGAGTA?CGGCCGCAAG?GCTAAAACTC 840
AAAGGAATTG?ACGGGGGCCC?GCACAAGCAG?CGGAGCATGT?GGCTTAATTC?GACGCAACGC 900
GAAGAACCTT?ACCAAGGCTT?GACATATGCC?GGAAACGTCT?AGAGATAGGC?GCCCCCTTGT 960
GGTCGGTATA?CAGGTGGTGC?ATGGTTGTCG?TCAGCTCGTG?TCGTGAGATG?TTGGGTTAAG 1020
TCCCGCAACG?AGCGCAACCC?TTGTTCTGTG?TTGCCAGCAT?GCCTTTCGGG?GTGATGGGGA 1080
CTCACAGGAG?ACTGCCGGGG?TCAACTCGGA?GGAAGGTGGG?GACGACGTCA?AATCATCATG 1120
CCCCTTATGT?CTTGGGCTGC?ACACGTGCTA?CAATGGTCGG?TACAAAGGGC?TGCGATGCCG 1180
CGAGGCGGAG?CGAATCCCAA?AAAGCCGGCC?TCAGTTCGGA?TTGGGGTCTG?CAACTCGACC 1240
CCATGAAGTT?GGAGTTGCTA?GTAATCGCAG?ATCAGCATGC?TGCGGTGAAT?ACGTTCCCGG 1300
GCCTTGTACA?CACCGCCCGT?CACGTCACGA?AAGTCGGTAA?CACCCGAAGC?CGGTGGCCTA 1360
ACCCGTAAGG?GGAGGAGCCG?TCGAAGGTGC?GG 1392
Claims (2)
1. a biological pesticide containing antibiotic is characterized in that effective ingredient is agricultural anti-HA97502, and molecular formula is C
25H
37O
4N, chemical structural formula is:
2. the preparation method of the described biological pesticide containing antibiotic of claim 1, the employing microbial fermentation technology is produced, it is characterized in that producing bacterium is A Bula streptomycete Streptomyces aburaviensis HA97502, and culture presevation is in CGMCC, and preserving number is CGMCC No.2297.
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|---|---|---|---|
| CN2008100608083A CN101300980B (en) | 2008-03-21 | 2008-03-21 | Biological pesticide containing antibiotic and preparation thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100608083A CN101300980B (en) | 2008-03-21 | 2008-03-21 | Biological pesticide containing antibiotic and preparation thereof |
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|---|---|
| CN101300980A CN101300980A (en) | 2008-11-12 |
| CN101300980B true CN101300980B (en) | 2010-04-21 |
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ID=40111232
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|---|---|---|---|
| CN2008100608083A Expired - Fee Related CN101300980B (en) | 2008-03-21 | 2008-03-21 | Biological pesticide containing antibiotic and preparation thereof |
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| CN101979630B (en) * | 2010-08-31 | 2012-11-21 | 浙江省农业科学院 | Method for preparing insecticidal bioactivator |
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Granted publication date: 20100421 Termination date: 20120321 |