CN1539957A - Hainan variety of lilac grey streptomycete and method for preparing agricultural antibiotic from variety - Google Patents
Hainan variety of lilac grey streptomycete and method for preparing agricultural antibiotic from variety Download PDFInfo
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Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
A Hainan variety of levendstreptomycete (CGMCC No.1026) is disclosed. An agricultural antibiotic for preventing and treating bacteria diseases of agricultural crops is prepared through the fermentation of levendstreptomycete's Hainan variety. Its advantages are high effect and low poison and residue.
Description
Technical field
The present invention relates to the novel bacterial of streptomyces and prepare the method for agricultural antibiotic by it.
Background technology
Agricultural chemicals is an important agricultural material, is bringing into play crucial effect in the high yield that guarantees farm crop, stable yields.At present, the annual pesticide preparation that uses of China closes former more than 30 ten thousand tons of medicines (pure) up to more than 100 ten thousand tons, and pesticide species is kind more than 200.Facing to huge agricultural chemicals demand and a large amount of inputs, China's pesticide industry has to have walked to copy external some pesticide species of crossing patent protection period, and this has caused the unreasonable of the backwardness of China's agricultural chemicals initiative and breed structure.Show 3 70% phenomenons highlightedly: sterilant accounts for 70% of agricultural chemicals, and severe toxicity and riskiest pesticide account for 70% of sterilant, and wherein the organophosphorus organochlorine pesticide accounts for 70% of high malicious highly toxic pesticide.
Microbial pesticide is often referred to the agricultural chemicals made from microorganism and meta-bolites thereof.Because China is vast in territory, weather condition, soil type, floristics etc. are varied, and this just provides good condition for the development of new microbial pesticide.
Summary of the invention
The novel bacterial that the purpose of this invention is to provide a kind of streptomyces, it can produce the new N-glucoside microbiotic of a class.This microbiotic has preventive and therapeutic effect to farm crop are bacillary with fungal disease.
Another object of the present invention provides by this novel bacterial and prepares such antibiotic method.
For achieving the above object, the present invention takes following scheme:
With Hainan mutation of lilac grey streptomycete, by the fermentor tank liquid submerged fermentation, fermented liquid contains a class glucoside compound, gives birth to plain bacterium during this compounds is called.The middle rhzomorph of giving birth to is to corps diseases, and particularly bacterial diseases of crops has tangible preventive and therapeutic effect.
Lilac grey streptomycete Hainan mutation of the present invention (Streptomyces lavendulae var.hainanensis) on October 29th, 2003 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preserving number is CGMCC No.1026.
CGMCC No.1026 is by the wild streptomycete of natural separation, obtains behind strain improvement.
CGMCC No.1026 has following microbiological property:
1. morphological characteristic
This bacterial classification can both form well-developed aerial mycelium and substrate mycelium on multiple classification substratum, the single wooden fork branch of mycelia forms curling volution fibrillae of spores 1-4 circle on the aerial mycelium top.Often form the conidia chain more than 10, spore circular or oval (0.6-0.8 * 1-1.2 μ m), spore surface is smooth.
2. cultivate and learn characteristic
This bacterial classification is planned on the substratum of drafting (being called for short the ISP substratum) at common classification substratum and international chain mould, form Powdered aerial mycelium, the grey of greyish white or micro mist, lilac grey are to dark violet ash, substrate mycelium creamy or light yellow to the mustard yellow, no soluble pigment.Can be referring to table 1.
The cultural characteristic of table 1 lilac grey streptomycete Hainan mutation
| Substratum | Aerial mycelium | Substrate mycelium | Soluble pigment |
| Yeast extract paste wort agar ISP2 | Powdered, light powder-lilac grey | Nothing-fermented bean drink Huang | Do not have |
| Oat agar ISP3 | Thin powdery, whitewash-light rose violet | Pale yellow-nude | Do not have |
| Inorganic salt starch ISP4 | Powdered, whitewash-lilac grey | Pale yellow-light cocoon Huang | Do not have |
| Glycerine Radix Asparagi agar ISP5 | Few, greyish white | Shallow honey is yellow | Do not have |
| Peptone-iron agar ISP6 | Baldness | Brown | Brown-dark brown |
| Tyrosine agar ISP7 | Powdered, white-greyish white | Pale yellow-the parched rice Huang | Do not have |
| Gao Shi synthesizes an agar | Powdered, whitewash-lilac grey | Pale yellow-nude | Do not have |
| Ke Shi synthesizes an agar | As thin as a wafer, little greyish white | Nothing-light cocoon Huang | Do not have |
| Cha Shi sucrose agar | Baldness | Milky white | Do not have |
| Bennett ' s agar | Powdered, white-micro mist | Light brown | Do not have-shallow brown |
| Emerson ' s agar | As thin as a wafer, little greyish white | Light brown | Do not have-shallow brown |
| Potato ball | Baldness | Light brown-brown | Shallow brown-brown |
3. physiological property
A. the melanic formation of class: on peptone-yeast extract paste-iron agar (ISP6), form brown pigment, but on tyrosine agar (ISP7), no soluble pigment.
B. gelatine liquefication: the positive.
C. milk is cultivated: milk solidifies and peptonizes.
D. starch hydrolysis: the positive.
E. nitrate reductase: the positive.
F. Mierocrystalline cellulose decomposes: do not decompose.
Hainan mutation of lilac grey streptomycete can utilize glucose, seminose, wood sugar, rhamnosyl, pectinose well.Next is fructose, inositol, synanthrin, lactose, semi-lactosi.Do not utilize sodium tartrate, N.F,USP MANNITOL, raffinose, sodium-acetate, Trisodium Citrate.Suspicious to utilizing of sucrose.
Middle living plain bacterium has 6 effective constituents, increases progressively a beta-lysine between each composition successively, and its structure is seen formula I.These 6 effective constituents are homologous compounds, and ply of paper is analysed the result and shown that it belongs to typical alkaline water dissolubility microbiotic monoid, and the ultra-violet absorption spectrum of mixture only shows end absorption, each component has similar infrared spectra, 3400,1720,1600,1560,1340 and 1050cm
-1Charateristic avsorption band is arranged, be all levorotatory compound.
Formula I
Wherein n is the positive integer of 1-6.
CGMCC No.1026 gives birth to the rhzomorph aqua in fermentation obtains step is as follows:
CGMCC No.1026 placed shake bottle and carry out seed culture, usual conditions are 28～30 ℃ of temperature, and shaking speed is 180～200rpm, cultivates 20～25 hours; In seeding tank, cultivated 24 hours; After fermenting 60～65 hours in the big jar of switching again, fermented liquid is carried out sheet frame or centrifuging; Filtrate is regulated pH value to 4.0-4.5, give birth to rhzomorph aqua finished product in making behind the last vacuum concentration.
Following summary of the invention partly is the positively effect of middle living rhzomorph:
A. give birth to plain bacterium in and the fungistatic effect of some common pathogen of crop is seen the 7th page table 2.MIC is minimum inhibition concentration (Minimal Inhibition Concentration).Middle application process and effect of giving birth to rhzomorph control corps diseases also can be referring to the 8th page table 3.
B. give birth to the toxicity of plain bacterium in to animal
(1) acute toxicity: big white mouse is oral, industrial goods LD
50Be 10000mg/kg, pure product LD
50Male is 3 16mg/kg, and female is 237mg/kg.Big white mouse is through skin, industrial goods LD
50＞10000mg/kg.
(2) pungency of skin and eyes: White Rabbit tuerculoderma not show spot forms, and no oedema forms.1～24h conjunctiva mild hyperaemia oedema after the medication of White Rabbit eyes, no secretory product, cornea is normal, and it is normal that 48h recovers.
(3) cumulative toxicity: always take the photograph sample size when equaling dead half by cumulative coefficient and remove medium lethal dose, middle living rhzomorph cumulative coefficient is 5.8.
(4) teratogenecity: the 6th day to the 15th day continuous irrigation stomach of rat gestation 10 days, dosage is 0.766～15.32mg/kg, each dosage is irritated the 0.5mg/100g body weight at every turn, and the embryo is not had influence.Through card square check, each dosage level absorbs the tire number and stillborn foetus number, tire mouse skeleton deformity and control group relatively do not have significant difference, and tire mouse internal organ are not seen deformity.
(5) micronucleus test: do not cause the effect of chromosome damage, do not cause the distored effect of sexual cell.
(6) mutagenicity: measure with the Ames dressing plate method of mixing, each dosage level does not all have mutagenesis.
C. give birth to the residual quantity of plain bacterium in plant materials, soil in
(1) specimen preparation: select the lotus Chinese cabbage ground of healthy growth, establish check plot and 30ppm spray medicine district, get and contrast soil sample, contrast dish sample, spray medicine soil sample, spray medicine dish sample, serial sampling 10 days every day behind the spray medicine.The dish sample is put tissue and is smashed to pieces in the ware and smash to pieces, soaks 24h, with gauze elimination dish slag.Filtrate is used the B vacuum filtration again with filter paper filtering twice.Filtrate is evaporated to a little volume, lyophilize in 50 ℃ of water-baths.Soil sample adds water breaks up, and behind the immersion 24h, uses filtered through gauze, filters with filter paper, B again, and filtrate is concentrated freeze-dried.
(2) sample determination: because middle living rhzomorph belongs to N-glucoside microbiotic, UV spectrum is an end absorption, general ultraviolet range can't detect, before having only the post of employing sample and o-phthalaldehyde(OPA) are carried out derivative reaction under proper condition, generation has the product of fluorescence, after separating with reversed phase ion pair chromatography again, detect with fluorimetric detector.Present method sensitivity is high, and minimum detectable activity is 6.9 * 10
-2Ppb, good reproducibility, relative standard deviation cv value is 0.2372%, the rate of recovery is 97.09%.
(3) measurement result: after the medication in the 1st day plant materials detected level be 0.185 μ g/ml, the 7th day detected level is 0.001 μ g/ml, do not detect in the 8th day, it is atomic at the occurring in nature remaining quantity to give birth to rhzomorph in this explanation, and the time only have 7 days, therefore, environment is not polluted, people and animals are not poisoned.
From above result as can be known, middle living rhzomorph is a kind of safe in utilization, the biological pesticide that is easy to decompose.
D. give birth to the farmland demonstration effect of plain bacterium in:
A.1991-1996 year gives birth to rhzomorph (15-30ppm) and prevents and treats test and the demonstration that bacterial blight of rice has been done system in Henan and two places, Beijing usefulness; comprise the not simultaneous test of with medicament; different onset degree and the test of control number of times; rice seedling bed dispenser protection is to the effect of land for growing field crops period of disease and occurring degree, and the experiment and demonstration area reaches 20,000 mu.The result shows that the best period that middle living rhzomorph waterproof is prevented and treated bacterial blight of rice is to soak seed, add rice seedling bed tri-leaf period again and transplant spraying in preceding 5 days.As the serious morbidity time, to spray 1-2 time again with the land for growing field crops their early stage, the expansion that can control the state of an illness spreads, and prevention effect is up to 86.3-90.1%, and it is strong to be better than chemical agent phenazine oxide and rice standing grain.
B.1992-2000 year is in Ruzhou, Henan, Zhengzhou, Puyang, replace chemical bactericides commonly used such as Tuzet with middle living rhzomorph in the orchard on ground such as the Yiyuan in Shandong, Yantai, prevent and treat ring rot of apple (decayed fruit disease) and leaf spot, living rhzomorph soup and the many liquid of ripple are used alternatingly among the employing 15-30ppm, spray altogether vegetative period and give birth to rhzomorph in 3 times, the decayed fruit rate generally can be controlled in 0.47-5%, quite or be better than chemical agent commonly used, give birth to the orchard of rhzomorph control in the use, fruit is big, color and luster good, fruit russeting also is one of outstanding feature less.
C.1992-2000 a year rhzomorph living in orchards, ground such as Zhengzhou, Puyang, Yiyuan are used is prevented and treated the apple leaf disease, and the sick leaf rate of leaf spot is controlled at about 22%, and disease index significantly is better than chemical agents such as Tuzet, derosal below 8%.
Advantage of the present invention: middle living rhzomorph can be prevented and treated corps diseases, particularly prevents and treats bacterial diseases of crops, applied range, and have the advantage of efficient, low toxicity, low residue.
Embodiment
The seed selection of embodiment 1:CGMCC No.1026
Lilac grey streptomycete Hainan mutation wild strain with natural separation obtains the big and full bacterial strain of a strain bacterium colony by natural seed selection, and color is a lavender.With this bacterial strain is starting strain, and its nearly 400 μ g/ml that tire obtain this bacterium protoplastis through the N,O-Diacetylmuramidase enzymolysis, use ultraviolet mutagenesis 15-50 second again, obtained mutagenesis effect preferably, obtain a strain CGMCC No.1026, it is tired up to 6000～7000 μ g/ml, is 17 times of starting strain.
Embodiment 2: the preparation of middle living rhzomorph aqua
CGMCC No.1026 placed shake bottle and carry out seed culture, condition is 28 ℃ of temperature, and shaking speed is 200rpm, cultivates 24 hours.Seed tank culture 24 hours, adopt the LM substratum in the seeding tank then.Seeding tank switching fermentation cylinder for fermentation was carried out sheet frame or centrifuging with fermented liquid after 60 hours.Filtrate is regulated pH value to 4.0, give birth to rhzomorph aqua finished product in making behind the last vacuum concentration (vacuum tightness 0.080MPa), effective component content is 10000 μ g/ml.
Cultivating the used shake-flask culture base of seed is (wt%): glucose 1.0, Zulkovsky starch 1.5, Semen Maydis powder 1.0, soybean cake powder 2.0, NH
4Cl 0.3, CaCO
30.3 and general trace element.Shaking bottled amount is the bottled 100ml of 500ml.
The seed tank culture base is (wt%): glucose 1.5, Semen Maydis powder 1.0, soybean cake powder 2.0, NH
4Cl0.4, CaCO
30.3, sodium-chlor 0.5 and general trace element.
Fermention medium is (wt%): glucose 2.5, Semen Maydis powder 1.0, soybean cake powder 2.0, NH
4Cl 0.4, CaCO
30.3, sodium-chlor 0.5 and general trace element.
Embodiment 3
CGMCC No.1026 placed shake bottle and carry out seed culture, condition is 30 ℃ of temperature, and shaking speed is 180rpm, cultivates 20 hours.Then seeding tank fermentation 24 hours, adopt the LM substratum identical in the seeding tank with embodiment 2.Seeding tank switching fermentation cylinder for fermentation was carried out sheet frame or centrifuging with fermented liquid after 65 hours.Filtrate is regulated pH value to 4.5, give birth to rhzomorph aqua finished product in making behind the last vacuum concentration (vacuum tightness 0.080MPa), effective component content is 9900 μ g/ml.
Give birth to the antimicrobial spectrum (MIC, the μ g/ml of unit) of rhzomorph different components in the table 2
| Test organisms | The n value | Fermented liquid (aqua) | |||||
| ????1 | ????2 | ????3 | ????4 | ????5 | ????6 | ||
| The soft rot of cabbage bacterium | ????50 | ????0.09～0.1 | ????1～2 | ????0.78 | ????0.39 | ????0.78 | ????1.56 |
| Withered germ of water-melon | ????25 | ????1.56 | ????0.78 | ????3～4 | ????0.78 | ????0.39 | ????0.78 |
| Pyricularia oryzae | ????12.5 | ????3～4 | ????0.39 | ????0.39 | ????0.1～0.2 | ????0.1～0.2 | ????3～4 |
| Aspergillus niger | ????50 | ????50 | ????0.78 | ????3～4 | ????1～2 | ????3～4 | ????12.5 |
| The white candiyeast | ????12.5 | ????100 | ????3～4 | ????0.78 | ????0.39 | ????0.39 | ????1～2 |
| Saccharomyces sake | ????25 | ????3～4 | ????0.78 | ????0.1～0.2 | ????3～4 | ????0.04 | ????1～2 |
| Bacillus proteus | ????50 | ????6～7 | ????3～4 | ????1～2 | ????1～2 | ????3～4 | ????6～7 |
| Intestinal bacteria | ????100 | ????50 | ????12.5 | ????6～7 | ????6～7 | ????12.5 | ????3～4 |
| Streptococcus aureus | ????3～4 | ????1～2 | ????0.1～0.2 | ????0.97 | ????0.09～0.1 | ????0.1～0.2 | ????0.39 |
| Sarcina | ????6～7 | ????25 | ????0.39 | ????0.97 | ????0.09～0.1 | ????0.1～0.2 | ????0.39 |
| Bacillus subtilus | ????6～7 | ????0.39 | ????0.09 | ????0.1～0.2 | ????0.04 | ????0.09～0.1 | ????0.1～0.2 |
| Mycobacterium tuberculosis | ????6～7 | ????1～2 | ????0.78 | ????0.39 | ????0.78 | ????1～2 | ????0.78 |
| Mycobacterium phlei | ????3～4 | ????0.1～0.2 | ????0.39 | ????0.9～1 | ????1～2 | ????0.39 | ????12.5 |
| Gibberella saubinetii | ????12.5 | ????0.097 | ????0.1～0.2 | ????0.39 | ????0.39 | ????0.39 | ????- |
| The rape black rot | ????50 | ????0.97 | ????3～4 | ????1～2 | ????1～2 | ????1～2 | ????- |
| The ginseng rust germ | ????6～7 | ????0.097 | ????1～2 | ????0.1～0.2 | ????0.1～0.2 | ????0.1～0.2 | ????- |
Give birth to rhzomorph control corps diseases and effect in the table 3
| Crop | Controlling object | Application process | Prevention effect (%) |
| Paddy rice | Bacterial leaf-blight | 100 μ g/ml seed soaking, 15-20 μ g/ml spraying 2-3 time | ????85-90 |
| Chinese cabbage | Soft rot | 50 μ g/ml seed soaking, 20-30 μ g/ml spraying 2-3 time | ????70-85 |
| Citrus | Peptic Ulcers | 20-30 μ g/ml spraying 4-5 time | ????70-85 |
| Tomato | Bacterial wilt | 20-30 μ g/ml irritates root 2-3 time | ????65-75 |
| Cucumber | Bacterial angular leaf spot | 20-30 μ g/ml spraying 1-2 time | ????80-90 |
| Ginger | Bacterial wilt of ginger | 50 μ g/ml seed soaking, 20-30 μ g/ml irritates root 2-3 time | ????70-80 |
| Reed | Stem wilt | 20-30 μ g/ml spraying 2-3 time | ????70-80 |
| Green pepper | Shot hole | 20-30 μ g/ml spraying 2-3 time | ????70-80 |
| Apple tree | Ring spot | 20-30 μ g/ml spraying 3-4 time | ????70-80 |
| Watermelon | Blight | 20-30 μ g/ml irritates root 2-3 time | ????60-75 |
Claims (7)
1. lilac grey streptomycete Hainan mutation, preserving number are CGMCC No.1026.
2. a compounds is characterized in that: have the structure of formula I,
Formula I
Wherein n is the positive integer of 1-6.
3. a microbiotic is characterized in that, is made up of in the described compound of claim 2 one or more.
4. the preparation method of the described antibiotic aqua of claim 3 is characterized in that, uses CGMCC No.1026 to carry out liquid submerged fermentation.
5. the preparation method of the described antibiotic aqua of claim 4 is characterized in that, CGMCC No.1026 is placed shake bottle and carry out seed culture, and condition is 28～30 ℃ of temperature, and shaking speed is 180～200rpm, cultivates 20～25 hours; In seeding tank, cultivated 24 hours; After fermenting 60～65 hours in the big jar of switching again, fermented liquid is carried out sheet frame or centrifuging; Filtrate is regulated pH value to 4.0-4.5, give birth to rhzomorph aqua finished product in making behind the last vacuum concentration.
6. the purposes of the described compound of claim 2 in crop disease control.
7. the purposes of the described compound of claim 2 in bacterial diseases of crops control.
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| TR01 | Transfer of patent right |
Effective date of registration: 20091113 Address after: No. 12 South Street, Beijing, Haidian District, Zhongguancun: 100081 Co-patentee after: FUJIAN KAILI BIO-PRODUCT Co.,Ltd. Patentee after: INSTITUTE OF ENVIRONMENT AND SUSTAINABLE DEVELOPMENT IN AGRICULTURE, CHINESE ACADEMY OF AGRICULTURAL SCIENCES Address before: No. 12 South Street, Beijing, Haidian District, Zhongguancun: 100081 Co-patentee before: Suzhou Kaili Biological Products Co.,Ltd. Patentee before: INSTITUTE OF ENVIRONMENT AND SUSTAINABLE DEVELOPMENT IN AGRICULTURE, CHINESE ACADEMY OF AGRICULTURAL SCIENCES |
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| CX01 | Expiry of patent term |
Granted publication date: 20050921 |
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| CX01 | Expiry of patent term |