CN101724576B

CN101724576B - Pyrethroid insecticide degradation bacteria and method for preparing fungicide thereof

Info

Publication number: CN101724576B
Application number: CN2008101199835A
Authority: CN
Inventors: 白志辉; 谷立坤; 唐玲; 荆梦; 庄国强; 张洪勋
Original assignee: Research Center for Eco Environmental Sciences of CAS
Current assignee: Research Center for Eco Environmental Sciences of CAS
Priority date: 2008-10-21
Filing date: 2008-10-21
Publication date: 2012-01-04
Anticipated expiration: 2028-10-21
Also published as: CN101724576A

Abstract

The invention relates to a pyrethroid insecticide degradation bacteria separated from Euonymus japonica phyllosphere and a method for preparing a fungicide thereof, belonging to the biotechnology field. By identification, the bacillus is Pseudomonas aeruginosa and is preserved in CCNCC with the preservation number of CGMCC No.2699. The method for producing the fungicide comprises five steps of preparing culture medium, activating strains, preparing liquid seeds, fermenting in a liquid state and preparing the fungicide. The pyrethroid insecticide degradation fungicide produced by the technical scheme of the invention can effectively degrade the pyrethroid insecticides on plant leave surfaces, reduce insecticide residual, ensure food safety and protect ecological environment.

Description

One pyrethroid insecticide degradation bacteria and bacterial preparation process thereof

Technical field

The present invention relates to the pyrethroids pesticide degradation bacterium that a strain separates arrogant leaf Chinese littleleaf box phyllosphere, and bacterial preparation process.Belong to biological technical field.

Technical background

Pyrethrin is a broad spectrum insecticide, has quick-acting, efficient, low toxicity, low residue, and to characteristics such as crop safeties, except that control has the special efficacy to 140 various pests, some pyrethrin also has the better prevention effect to underground insect and mite pest.Chinese patent (Granted publication number: CN1063017C) announced that a kind of Avermectin, pyrethroid (fenvalerate, PP-383) are the compounded pesticides of effective constituent, be mainly used in the control small cabbage moth, Agricultural pests such as prodenia litura.Chinese patent (Granted publication number: CN1242675C) announced a kind of with Avrmectin and beta_cypermethrin as major ingredient, add emulsifying agent, the insecticides that mixed organic solvents and water are made into can be prevented and treated insects such as pear sucker, liriomyza bryoniae.

As efficient, low toxic pesticide, pyrethroid is mainly used in Agricultural pests and the sanitary insect pest on control cotton field, vegetable plot, fruit tree and the tealeaves.Its usage quantity was increasing in recent years, and pyrethroid has become one of substitute of traditional organophosphorus pesticide, but found in recent years that it was bigger to aquatic ecosystem harm.As Wang Yanmei etc. (lambda-cyhalothrin is to the acute toxicity effect of swamp eel, the Anhui agricultural sciences, 2008,36 (12): though 4999-5000) during report 48h the lambda-cyhalothrin swamp eel of 0.0552 μ g/L all dead.

Carried out some researchs to pyrethrin is residual both at home and abroad, mainly contain agricultural chemicals use and residual, reduce degradation rule in plant materials and in the environment of the residual countermeasure of farming, agricultural chemicals, the research of degradation of pesticide mikrobe etc.But, for how reducing the residual research of farming only limiting to rationally use agricultural chemicals, improve agronomic measures, strengthen management etc., concrete measure is perfect inadequately, lacks the directly effective ways of reduction target pesticide residue; In addition, the research to pesticide degradation bacteria is confined to the separation of bacterial strain and the mensuration of physiological property thereof more.

(separation and the evaluation of a strain PP-383 degradation bacteria, Agricultural University Of Nanjing's journal 2007,30 (3): 68-72) from the PP-383 contaminated soil, be separated to a strain degradable PP-383 and bifenthrin Pseudomonas aeruginosa such as Liu Junhan.Hong Yongcong etc. publish an article, and (bacillus cereus strain TR2 is to the plot experiment of PP-383 Degradation; Qingdao Agricultural University's journal (natural science edition) 2008; 24 (4): 291-293) point out test through indoor pot and sub-district, tea place, bacillus cereus strain TR2 can effectively reduce the residual of sub-district, tea place PP-383.

At present, do not find have bibliographical information to isolate the degradation bacteria and the bacterial preparation process thereof of pyrethrin from plant phyllospheric as yet.

Summary of the invention

The purpose of this invention is to provide the pyrethrin degradation bacteria that a strain separates arrogant leaf Chinese littleleaf box phyllosphere, and bacterial preparation process.

Biocontrol strain provided by the present invention is to be come out by our laboratory separation screening from the blade face of Euonymus japonicus.It is numbered EBL-07, is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date is on October 13,2008, classification called after Pseudomonas aeruginosa (Pseudomonas aeruginosa).Deposit number is: CGMCC No.2699.This bacterial strain has following character:

Colony morphology characteristic and morphological features

Bacterium colony is Vandyke brown, flat, irregular, edge spination on the LB solid medium.Be blackish green in the LB liquid nutrient medium, no gemma, gramstaining is negative, and oxydase and catalase are all the positive, and the V.P reaction is negative, and the hydrolyzable gelatin can move.

Resistance is measured

Bacterial strain shows 6 kinds of antibiotic sensitivity testing results: bacterial strain has resistance to kantlex, penbritin, to many glutinosins, paraxin, tsiklomitsin, qingfengmeisu qiong sensitivity.

16S rDNA gene order

Use the partial sequence of the 16S rDNA gene of this bacterial strain that 27f and 1492r amplify as primer, sequencing result is delivered to GenBank, and accession number is EF545556.On http://www.ncbi.nlm.nih.gov/ website, compare with blast program and the 16S rDNA gene order of landing bacterial isolates, the result shows with the similarity of Paenibacillus polymyxa the highest, reaches more than 99%.

The working method of microbial inoculum of the present invention is following:

1, substratum preparation: a) the LB substratum (solid, 1L): peptone 10g, yeast extract paste 5g, sodium-chlor 5g, agar 20g adds water to 1L, pH7.0; B) the LB substratum (liquid, 1L): peptone 10g, yeast extract paste 5g, sodium-chlor 5g adds water to 1L, pH7.0; C) seed culture medium (liquid, 1L): MgSO ₄0.10g, KH ₂PO ₄0.50g, NaCl0.50g, K ₂HPO ₄1.50g, sucrose 10g, peptone 5g adds water to 1L, pH7.0; D) fermention medium (liquid, 1L): MgSO ₄0.10g, KH ₂PO ₄0.50g, NaCl0.50g, K ₂HPO ₄1.50g, sucrose 10g, steeping water 5g adds water to 1L, pH7.0; Above substratum is all at 121 ℃ of sterilization 20min.

2, actication of culture: picking pyrethroid degradation bacteria is to the LB solid medium, and after 28 ℃ line was chosen single bacterium colony and cultivated twice continuously, picking list bacterium colony was at the liquid substratum of LB, in 28 ℃, and 150r/min shaking table shaking culture overnight cultures;

3, liquid seeds preparation: in the seed culture medium of high-temperature sterilization, according to the inoculum size inoculation pyrethroid degradation bacteria of 5%-10%, in 25-35 ℃, 200r/min shaking table shaking culture 12-24h obtains liquid seeds.

4, liquid state fermentation: in autoclaved seed culture medium, inoculation pyrethroid degradation bacteria, in 25-35 ℃, 200r/min shaking table shaking culture 18-72h.

5, fungicide preparation: cultured fermented liquid is adjusted to 10 after bacterial count ⁸-10 ¹⁰Cell/mL adds glycocoll (massfraction 0.5%), and packing is used.

The pyrethroid degradation bacterial agent of being produced by the technical scheme of the present invention blade pyrethroid of can effectively degrading is residual.Below specify enforcement of the present invention through specific embodiment, purpose is to help the reader to understand spirit of the present invention better, but not as to the qualification of practical range of the present invention.

Embodiment 1: the preparation of pyrethroid degradation bacterial agent

1) substratum preparation: a) the LB substratum (solid, 1L): peptone 10g, yeast extract paste 5g, sodium-chlor 5g, agar 20g adds water to 1L, pH7.0; B) the LB substratum (liquid, 1L): peptone 10g, yeast extract paste 5g, sodium-chlor 5g adds water to 1L, pH7.0; C) seed culture medium (liquid, 1L): MgSO ₄0.10g, KH ₂PO ₄0.50g, NaCl0.50g, K ₂HPO ₄1.50g, sucrose 10g, peptone 5g adds water to 1L, pH7.0; D) fermention medium (liquid, 1L): MgSO ₄0.10g, KH ₂PO ₄0.50g, NaCl0.50g, K ₂HPO ₄1.50g, sucrose 10g, steeping water 5g adds water to 1L, pH7.0; Above substratum is all at 121 ℃ of sterilization 20min.

2) bacterial strain activation: picking pyrethroid degradation bacteria is to the LB solid medium, and after 28 ℃ line was chosen single bacterium colony and cultivated twice continuously, picking list bacterium colony was at the liquid substratum of LB, in 28 ℃, and 150r/min shaking table shaking culture overnight cultures;

3) liquid seeds preparation: in the seed culture medium of high-temperature sterilization, inoculation pyrethroid degradation bacteria, in 30 ℃, 200r/min shaking table shaking culture 20h obtains liquid seeds.

4) liquid state fermentation: in autoclaved seed culture medium, inoculation pyrethroid degradation bacteria, in 30 ℃, 200r/min shaking table shaking culture 24h.

5) cultured fermented liquid is adjusted to 10 after bacterial count ¹⁰Cell/mL adds glycocoll (massfraction 0.5%), and packing is used.

Embodiment 2: the pyrethroid degradation bacterial agent is to Euonymus japonicus blade face beta-cypermethrin degrading effect

1) chooses the Euonymus japonicus of non-dosed beta_cypermethrin, choose two plot, each 15m ², spraying amount of application is 3g/ mu;

2) behind the 6h, even spraying pesticide degradation bacterium preparation 100mL (10 ⁹Cfu/mL), compare to spray clear water;

3) behind the 2d, it is residual that five point samplings detect Euonymus japonicus blade face beta_cypermethrin, finds that treatment group reduces 76% than control group beta_cypermethrin residual quantity.

Embodiment 3: the pyrethroid degradation bacterial agent is to rape blade face FCR-1272 degradation effect

4) choose the rape of non-dosed FCR-1272, choose two plot, each 15m ², spraying amount of application is 1g/ mu;

5) behind the 6h, even spraying pesticide degradation bacterium preparation 100mL (10 ⁹Cfu/mL), compare to spray clear water;

6) behind the 2d, five point samplings detect the residual of rape blade face FCR-1272, find that treatment group reduces 83% than control group FCR-1272 residual quantity.

Claims

1. the bacterium that a strain can the efficient degradation pyrethroid pesticide; It is characterized in that: this bacterium is a Pseudomonas aeruginosa strain (Pseudomonas aeruginosa); Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number: CGMCC No.2699.

2. utilize the said bacterium of claim 1 to prepare the preparation method of pyrethroid degradation bacterial agent, it is characterized in that: the preparation method of microbial inoculum comprises the steps:

(1) substratum preparation: a) LB solid medium: peptone 10g, yeast extract paste 5g, sodium-chlor 5g, agar 20g adds water to 1L, and pH 7.0; B) LB liquid nutrient medium: peptone 10g, yeast extract paste 5g, sodium-chlor 5g adds water to 1L, and pH 7.0; C) liquid seed culture medium: MgSO ₄0.10g, KH ₂PO ₄0.50g, NaCl 0.50g, K ₂HPO ₄1.50g, sucrose 10g, peptone 5g adds water to 1L, and pH 7.0; D) liquid fermentation medium: MgSO ₄0.10g, KH ₂PO ₄0.50g, NaCl 0.50g, K ₂HPO ₄1 .50g, sucrose 10g, steeping water 5g adds water to 1L, and pH 7.0; Above substratum is all at 121 ℃ of sterilization 20min;

(2) actication of culture: picking pyrethroid degradation bacteria is to the LB solid medium, and after 28 ℃ line was chosen single bacterium colony and cultivated twice continuously, picking list bacterium colony was at the liquid substratum of LB, and in 28 ℃, 150r/min shaking table shaking culture is spent the night;

(3) liquid seeds preparation: in the seed culture medium of high-temperature sterilization, according to 5%～10% inoculum size inoculation pyrethroid degradation bacteria, in 25～35 ℃, 200r/min shaking table shaking culture 12～24h obtains liquid seeds;

(4) liquid state fermentation: in autoclaved seed culture medium, inoculation pyrethroid degradation bacteria, in 25～35 ℃, 200r/min shaking table shaking culture 18～72h;

(5) fungicide preparation: cultured fermented liquid is adjusted to 10 after bacterial count ⁸-10 ¹⁰Cell/mL, the glycocoll of adding massfraction 0.5%, packing.