CN1212389C - Antibiotic streptomyces and its preparation method - Google Patents

Abstract

The present invention discloses a new antibiotic streptomycin and a preparation method thereof. The antibiotic streptomycin is Streptomycesdaliensis sp. nov. CCTCC NO. 201031. Soil samples are dried and preprocessed, are diluted by sterilized water, are spread on isolation culture media, and are inversely cultured in a thermotank. Single bacterial groups are selected, and are cultured on a culture medium. Pure bacterial strains separated are fermented in a shake flask. The water phase samples and the fatty phase samples of fermented liquid are used for biological sieving. Then, the water phase samples, artificial diet and target insect egg blocks are mixed and cultured. The new antibiotic streptomycin has the advantages of high speed, high sensitivity, small sample amount, high repeatability, high insecticidal activity, wide insecticidal spectrums, high safety, and good effect on cotton bollworms, beet armyworms, cabbage worms, diamondback moths and pine caterpillars.

Description

A kind of antibiotic streptomyces and preparation method

Technical field

The present invention relates to have the antibiotic streptomyces (Streptomycs daliensissp.nov.) of insecticidal activity, also relate to the preparation method of this streptomyces antibioticus.

Background technology

Along with the widespread use of chemical pesticide in crop pest control, environmental pollution and public hazards that it caused also are on the rise.And in the residual food chain that causes of chemical pesticide agricultural chemicals accumulate waywardly, caused a large amount of drug-induced diseases, reduced people's quality of life, damaged the people's health.Aspect Agricultural Export earned foreign exchange, pesticide residue had also reduced the competitive capacity of China's agricultural byproducts.The use of simultaneously a large amount of chemical pesticides causes the part harmful organism resistance to occur, not only increases the enforcement difficulty of the comprehensive regulation, and seriously hinders the development of sustainable agriculture.The serious consequence that above-mentioned chemical pesticide brought widely common people is paid close attention to, and also causes national governments and scientist's great attention.Nineteen ninety, EPA has been cancelled the registration to 59 kinds of chemical pesticides, and European Union has formulated the strategic plan that reduces chemical pesticide, the inaccurate chemical pesticide that uses of garden crops such as vegetables that some national regulations are directly edible.1992, the World Food Programme held the biological pesticide world congress, proposed biological pesticide and will account for 60% of 21st century agricultural chemicals sales volume.Therefore the nineties in 20th century whole world chemical pesticide sale constantly descend, and can be mass-produced, efficient, non-harmful microbial pesticide develops rapidly and occupies increasing share.The Chinese government has classified biological pesticide as the preference of " China Agenda 21 ".

Microbial pesticide is the sterilant that derives from microorganism, divides self and meta-bolites two big classes, belongs to natural product.The former is as Bacillus thuringiensis, virus, fungi, nematode, the latter such as Avermectin (Avrmectin), Tracer (dish happiness) etc.The Biotrol BTV pest control is obtained very ten-strike, and the remarkable effect of insects such as Avrmectin control liriomyza bryoniae also causes extensive concern.1997, U.S. DowElanco company released the Tracer that is produced by actinomycetes, and this product toxicity is low, the insecticidal activity height, and the desinsection spectrum width is described as the cookle of field of pesticides, and another has shown the huge power of microbial pesticide.

At present, from the microbial secondary meta-bolites, screened nearly ten thousand kinds of biologically active substances such as microbiotic, reaching about hundred kinds of mass production and widespread use wherein, economic benefit is considerable.But wherein 60% produce by actinomycetes.Antiinsect antibiotic that screens from actinomycetes such as macro tetrolide (Polynactin), the fundamental research of Avrmectin (Avermectin) etc. and commercialized development have all been obtained and have been made us seeing the purpose achievement.

Biogenic pesticide has obtained comprehensive exploitation and has used widely, but its research, exploitation, production and application all also exist some distinct issues and difficulty, are stranded the development of scratching China's biogenic pesticide.It is slow to knock down speed as biogenic pesticide, and validity period is short, and insecticidal spectrum is narrower, not strong to the pest population control in a short time relatively, can not reduce the insect population density rapidly.Relatively insecticidal spectrum is narrower, is unfavorable for that a medicine controls more, and as when various pests appears in vegetable field, just attend to one thing and lose sight of another, unable to do what one wishes, there is certain difficulty in application.

Summary of the invention

The object of the present invention is to provide a kind of antibiotic streptomyces with insecticidal activity, isolating strain actinomycetes from soil, ferment with substratum, fermented liquid and extract thereof are carried out bioactivity screening by bollworm and two kinds of insect pest models of beet armyworm.This bacterial strain fat extracts the sample beet armyworm mutually very strong killing effect, insecticidal activity height, desinsection spectrum width, safety, the microbial pesticide of low toxicity.

Another object of the present invention is to provide a kind of preparation method who prepares antibiotic streptomyces.

In order to achieve the above object, the present invention has adopted following technical measures:

Will be from soil isolating strain actinomycetes, ferment with two kinds of substratum respectively, fermented liquid and extract thereof are carried out bioactivity screening by bollworm and two kinds of insect pest models of beet armyworm, and bollworm and beet armyworm all belong to the lepidopteran Noctuidae, the polyphagy insect.The water that studies have shown that this bacterial strain extracts sample mutually with fat has very strong killing effect to beet armyworm, faint to the bollworm effect; Its fat extracts sample mutually also to cabbage caterpillar, small cabbage moth, the certain insecticidal activity of pine moth performance.Therefore the fermented sample of this bacterium can be used for guaranteeing the important leverage of agricultural production and income and Sustainable development in the integrated control of harmful organism of farm crop and forest, and with improve species diversity and protection environment, guarantee that human and livestock health is closely bound up.In conjunction with phenotypic classification (form and physiological and biochemical property), several different methods such as chemical classification and molecular classification is carried out polyphase sort to these strain actinomycetes, the result shows the novel species of this Pseudomonas in streptomyces, because of these actinomycetes are to separate from the soil that gather in Dali, so with its called after Dali streptomycete (this bacterial strain of Streptomyces daliensis sp.nov. is in China's typical culture collection center preservation, and preserving number is CCTCC NO.M201031).

The steps include:

1, with the soil sample of being gathered through 120 ℃ of xeothermic pre-treatment 1 hour, can reduce fungi, number of bacteria, increase the actinomycetic occurrence rate of spore.

2, will be diluted to finite concentration with sterilized water through pretreated soil sample, and be coated on isolation medium 1 and the isolation medium 2, and be inverted in 28 ℃ of thermostat containers and cultivate, 3-7 day is selected single bacterium colony, cultivates on No. 38 substratum.

3, isolating pure bacterial strain carries out shake flask fermentation with KN-1 and KN-2 substratum respectively, and the water of fermented liquid and fat sample mutually all are used for biological screening, so just can not cause the leakage sieve of active substance.

4, fermented liquid is aqueous sample, and fermented liquid is a fat phase sample with the sample of ethyl acetate extraction.

5, with aqueous sample and artificial diet, target insect pieces of an egg mixed culture, respectively at 1 day, 2 days, hatching situation, the size of polypide and the death condition of insect of 4 days observation pieces of an egg.

6, with fat phase sample (0.6 milliliter) with 100 microlitre dimethyl sulfoxide (DMSO) (DMSO) dissolving back with 4900 microlitre distilled water dilutings as life test sample product, the 100 microlitre test sample product of getting mix with examination worm artificial diet, pieces of an egg or the newly hatched larvae of target insect are put into wherein, observe reaction symptom therebetween, incubate the piece hatching, survival for the examination insect, get food, individual size etc., and give birth to respectively and survey the size of checking the mortality ratio and the worm that lives after 4 days, and with compare.

7, the activated bacterial strain of sample has carried out form and physiological and biochemical property, the research of cell walls chemical composition.16SrDNA sequence according to this bacterial strain is carried out molecular classification.The 16SrDNA of this bacterial strain measures 1489 effective bases altogether, and the sequence number of applying at GenBank is AY029696.Phylogeny data and grouped data (uncle Jie Shi bacterium handbook) can determine that this bacterial strain is a novel species of streptocin, because of this bacterial strain is to separate to obtain called after Dali streptomycete (Streptomyces daliensis sp.nov.) from the soil sample that gather in Dali

8, screening model.

The character of Dali streptomycete

According to above-mentioned implementation method, obtaining a strain has the actinomycetes of insecticidal activity, be accredited as the novel species of streptomyces through polyphase sort, called after Dali streptomycete (Streptomyces daliensis sp.nov.), this bacterial strain in August 30 calendar year 2001 in China's typical culture collection center preservation, preserving number is CCTCC NO.M201031.

1. the insecticidal activity of Dali streptomycete: this bacterial strain is cultivated the water that obtains and fat phase sample to beet armyworm (Spodoptera exiaua with two kinds of fermentation culture, be abbreviated as Se) very strong killing effect arranged, to bollworm (Helicoverpa armigera, be abbreviated as Ha) effect faint, and the result is stable, good reproducibility, the result is as follows.

Sample type	Substratum	Give birth to and survey the date	Target	Killing rate (%)
					Water	KN-1	2000-9-12	Se	100

Water	KN-2	2000-9-12	Se	90
					Water	KN-2	2000-9-12	Se	90
Water	KN-2	2000-10-16	Se	100
					Water	KN-2	2000-10-18	Se	100
Water	KN-1	2000-10-18	Se	100
					Water	KN-1	2000-10-19	Se	100
The fat phase	KN-2	2000-10-20	Se	90
					The fat phase	KN-1	2000-10-20	Se	100
Water	KN-1	2000-10-23	Se	100
					The fat phase	KN-1	2000-10-27	Se	100
The fat phase	KN-2	2000-10-27	Ha	40
					The fat phase	KN-1	2000-10-28	Se	100
The fat phase	KN-2	2000-10-28	Se	100
					The fat phase	KN-1	2000-10-28	Ha	30
The fat phase	KN-2	2000-10-30	Ha	40
					The fat phase	KN-1	2000-10-30	Ha	40
The fat phase	KN-2	2000-10-31	Se	95
					Water	KN-1	2000-11-8	Se	95
Water	KN-1	2000-11-8	Ha	30

2. the character of Dali streptomycete

(1) morphological specificity: bacterial strain buries the sheet cultivation after 15 days, with form and the spore shape that opticmicroscope and electron microscope are observed mycelia respectively, surface tissue with the ISP5 substratum.The result shows that the base silk grows good, and multi-branched does not form tabula and do not rupture yet.The growth of gas silk is vigorous, and long spore chain is arranged on the gas silk, is lax thin spiral.The spore chain length is divided into spore, and (0.6-0.8 * 0.4-0.6um), spore surface is smooth, and fold is arranged to ellipse for the spore oval.

(2) in the feature on the various substratum: after 28 ℃ of cultivation inoculations, observed three times respectively at the 7th, 15,30 day, with the standard of " The ISCC-NBS COLOR CHARTS Standard samples NO 2106 " (Kelly, 1964) chromatogram as color description.

1. Czapek's agar (Czapeks agar)

Aerial hyphae grows, grey yellowish brown

The vegetative hyphae canescence

Soluble pigment does not have

2. glycerine-amino-succinamic acid agar (ISP NO5)

The aerial hyphae growth is medium, white

The dark-grey yellow of vegetative hyphae

Soluble pigment does not have

3. inorganic salt-Starch Agar (ISP NO4)

Aerial hyphae grows, grey yellowish brown

The vegetative hyphae canescence

Soluble pigment does not have

4. yeast extract paste-malt extract agar (ISP NO2)

Aerial hyphae grows, canescence

Yellow in the vegetative hyphae

Yellow in the soluble pigment

5. rolled oats agar (ISP NO3)

Aerial hyphae grows, grayish brown

Vegetative hyphae ash yellow-green colour

Soluble pigment does not have

6. potato is soaked fat agar

Aerial hyphae grows, shallow taupe

The dark-grey yellow of vegetative hyphae

Soluble pigment does not have

(3) physiological and biochemical property:

1. gelatine liquefication+(the black brown pigments is arranged)

2. milk solidify-

Milk peptonizes-

3. nitrate reduction-

4. hydrogen sulfide generation-

5. pigment generation+

(4) utilization of carbon source

Fine utilization: D-fructose, D-pectinose, sucrose, D-wood sugar, D-inositol and glucose

Do not utilize: L-rhamnosyl, raffinose and N.F,USP MANNITOL

(5) cytochemistry component

Contain LL-DAP and a small amount of meso-DAP in the pure cell walls, and contain Asp and Gly; Its full cell wall hydrolysate contains semi-lactosi, glucose; A small amount of wood sugar, ribose, pectinose.

(6) 16S rDNA sequence

1. sequencing kit: Big Dye ^TMTerminator Cycle Sequencing Ready ReactionKit (PERKIN ELMER);

2. sequencing analysis instrument: ABI PRIS ^TM377 DNA Sequencer.

3. sequencing primer:

KMS098PBlr(5’-TAAGGAGGTGATCCAGCC-3’)；

KMS098PDr(5’-TAAGGAGGTGATCCAGCC-3’)；

KMS098PDr(5’-GGGTTGCGCTCGTTG-3’)；

KMS098PCr(5’-TCTGCGCATTTCACCGCTAC-3’)。

4. sequence amounts to 1489bp

ccggtcgacgagctcagagtttgatcctggctcaggacgaacgctggcggcgtgcttaaca

catgcaagtcgaacgatgaagcccttcggggtggattagtggcgaacgggtgagtaacacg

tgggcaatctgcccttcactctgggacaagccctggaaacggggtctaataccggatacga

gcttccaaggcatcttggaggttggaaagctccggcggtgaaggatgagcccgcggcctat

cagcttgttggtgaggtaatggctcaccaaggcgacgacgggtagccggcctgagagggcg

accggccacactgggactgagacacggcccagactcctacgggaggcagcagtggggaata

ttgcacaatgggcgaaagcctgatgcagcgacgccgcgtgagggatgacggccttcgggtt

gtaaacctctttcagcagggaagaagcgaaagtgacggtacctgcagaagaagcgccggct

aactacgtgccagcagccgcggtaatacgtagggcgcaagcgttgtccggaattattgggc

gtaaagagctcgtaggcggcttgtcacgtcgattgtgaaagcccgaggcttaacctcgggt

ctgcagtcgatacgggctagctagagtgtggtaggggagatcggaattcctggtgtagcgg

tgaaatgcgcagatatcaggaggaacaccggtggcgaaggcggatctctgggccattactg

acgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacgccgt

aaacggtgggaactaggtgttggcgacattccacgtcgtcggtgccgcagctaacgcatta

agttccccgcctggggagtacggccgcaaggctaaaactcaaaggaattgacgggggcccg

cacaagcggcggagcatgtggcttaattcgacgcaacgcgaagaaccttaccaaggcttga

catacaccggaaacgtctggagacaggcgcccccttgtggtcggtgtacaggtggtgcatg

gctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgt

tctgtgttgccagcatgcccttcggggtgatggggactcacaggagaccgccggggtcaac

tcggaggaaggtggggacgacgtcaagtcatcatgccccttatgtcttgggctgaacacgt

gctacaatggccggtacaatgagctgcgataccgtgaggtggagcgaatctcaaaaagccg

gtctcagttcggattggggtctgcaactcgaccccatgaagtcggagtcgctagtaatcgc

agatcagcattgctgcggtgaatacgttcccgggccttgtacacaccgcccgtcacgtcac

gaaagtcggtaacacccgaagccggtggcccaacccttgtggagggagctgtcgaaggtgg

gactggcgattggcacgaagtcgta

The present invention compared with prior art has the following advantages and effect:

1, She Ji biological screening method has fast, sensitivity, and sample size is little, and advantages such as good reproducibility are suitable for extensive primary dcreening operation;

2, this research lepidopterous two kinds of main harm insects of selection (bollworm and beet armyworm) are target insect, have the importance on the agricultural, and existing economic implications also has the market requirement, help product exploitation, production and popularization in the future;

3, this biological screening model will be given birth to test sample product and artificial diet, target insect pieces of an egg mixed culture, respectively at 1 day, 2 days, observed in 4 days, can obtain killing ovum, desinsection, suppress, avoiding multiple virulence data and virulence information such as keep away of sample, the feedback information of these biological assays is to the design and the composition optimizes of pesticide molecule, and the reasonableness biological pesticide of promotion being developed new mechanism of action has directive significance;

4, biological screening directly is material with the live body insect, and is more directly perceived than isolated measuring method, more approaching reality.

5, the fat phase extract of Dali streptomycete all shows certain insecticidal activity to bollworm, beet armyworm, cabbage caterpillar, small cabbage moth, pine moth, to beet armyworm, cabbage caterpillar, pine moth effect efficiently.

Embodiment

1. the separation of bacterial classification and cultivation

Before separating with soil sample with 120 ℃ of high temperature dry heat treatment one hour, adopt two kinds of separation methods to carry out pre-treatment then: 1) 6% yeast decoction added in the 5umol phosphoric acid buffer, 40 ℃ of processing 20 minutes; 2) use 6% yeast decoction, 0.1%SDS, 2mgNA (Nalidixic Acid) handles.Isolation medium adopts nutrient agar medium, adds 20ppmNA or 50ppm potassium bichromate respectively as selecting inhibitor, 28 ℃ of cultivations, selects the free of contamination single bacterium colony of specific form 3-7 day, cultivates on No. 38 substratum at last.

(1) isolation medium 1: Zulkovsky starch 1%, casein 0.03%, saltpetre 0.2%, 7 water magnesium sulfate 0.005%, sodium-chlor 20%, dipotassium hydrogen phosphate 0.2%, lime carbonate 0.002%, ferric sulfate 0.001%, water 78%, pH7.0; 28 ℃ of culture temperature.

(2) isolation medium 2 (ISP5): L-asparagine 0.1%, glycerine 1%, dipotassium hydrogen phosphate 0.1%, 1 milliliter of trace salt solution, vitamin complex 0.000375%, sodium-chlor 15%, water 84%, pH=7.0-7.4,28 ℃ of culture temperature.

(3) No. 38 substratum: yeast extract 0.4%, glucose 0.4%, malt extract 0.5%, vitamins B ₁0.0001%, vitamins B ₆0.0001%, riboflavin 0.0001%, nicotinic acid 0.0001%, phenylalanine 0.0001%, vitamin H 0.0001%, L-Ala 0.03%, water 98.7%, pH7.2,28 ℃ of culture temperature.

2. the shake flask fermentation of bacterial classification

Isolating pure bacterial strain is carried out shake flask fermentation with KN-1 and KN-2 substratum respectively, and shaking bottled amount is 100ml, and shaking speed is 100rpm, 28 ℃ of culture temperature, incubation time is 7 days, the microscopic examination of sampling smear, and pollution-free and fermented liquid that strain growth is good is used for extracting.

(1) shake flask fermentation substratum KN-1: dregs of beans 2%, peptone 0.2%, glucose 2%, starch 0.5%, yeast extract paste 0.2%, dipotassium hydrogen phosphate 0.05%, sal epsom 0.05%, lime carbonate 0.2%, sodium-chlor 0.4%, pH7.8.

(2) shake flask fermentation is amassed wealth by heavy taxation and is supported basic KN-2: glucose 1%, Zulkovsky starch 1%, dregs of beans 2.5%, yeast extract paste 0.3%, sodium-chlor 0.2%, lime carbonate 0.5%, pH7.0.

3. the extraction of fermented liquid

Get 5ml fermented liquid (hydroaropic substance) and be used for biological assay; Remaining fermented liquid all moves in the centrifuge tube of 50ml volume, centrifugal 10 minutes of 5000rpm, abandon supernatant, lower floor's mycelium is used the homogenate of cell stamp mill after adding the 25ml ethyl acetate, sealing orifice soaks under the room temperature that 5000rpm made it layering in centrifugal 10 minutes after 12 hours, and organic phase (upper strata) is moved in the 50ml glass cylinder, evaporation of acetic acid ethyl ester in vacuum drying oven adds 5ml methyl alcohol again and makes the extract dissolving.Get 0.6ml dissolve with methanol liquid in the Eppendorf pipe, all the other 4.4ml lysates move into dry in the lump evaporation methyl alcohol in the 5ml centrifuge tube, and the sample in the Eppendorf pipe (lyophobic dust) is used for biological assay, and the sample in the centrifuge tube is in-20 ℃ of storages.

4. biological assay

With the hydrophobicity sample with dimethyl sulfoxide (DMSO) (DMSO) dissolving back with distilled water diluting to finite concentration as giving birth to test sample product, wetting ability sample (fermented liquid) is directly as living test sample product.Test sample product to be generated are mixed with examination worm artificial diet, pieces of an egg or the newly hatched larvae of a certain amount of target insect are put into wherein, observe reaction symptom (pieces of an egg hatching therebetween for the examination insect, survival, get food, individual size etc.), and check mortality ratio and the size of the worm that lives after 4 days respectively at giving birth to survey, and with compare.

Claims (1)

1, a kind of antibiotic streptomyces is characterized in that it is antibiotic streptomyces (Streptomycesdaliensis sp.nov.), CCTCC NO.M201031.