CN105420172A - Metribuzin pesticide residue degrading bacteria, microbial agent produced through same and application of metribuzin pesticide residue degrading bacteria - Google Patents
Metribuzin pesticide residue degrading bacteria, microbial agent produced through same and application of metribuzin pesticide residue degrading bacteria Download PDFInfo
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- CN105420172A CN105420172A CN201610013489.5A CN201610013489A CN105420172A CN 105420172 A CN105420172 A CN 105420172A CN 201610013489 A CN201610013489 A CN 201610013489A CN 105420172 A CN105420172 A CN 105420172A
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- A62—LIFE-SAVING; FIRE-FIGHTING
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- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
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Abstract
The invention provides metribuzin pesticide residue degrading bacteria and a microbial agent produced through the same. The bacterial strain is gram-positive bacteria, and is authenticated as Rhodococcus sp. and preserved in the China Center for Type Culture Collection on June 29, 2015, and the preservation number of the bacterial strain is CCTCC NO: M2015392. A culture solution obtained after QCT10 bacterial strain fermentation is the microbial agent. According to the QCT10 bacterial strain and the microbial agent produced through the same, the quantity of metribuzin pesticide residues in soil can be reduced by 50% or above, and the problem that the metribuzin pesticide residues exceed the standard in agricultural production can be solved.
Description
Technical field
The invention belongs to biological high-tech field, microbial inoculum and the application of a kind of piperazine humulone pesticide residual degrading bacteria and production thereof are provided.
Background technology
The use of chemical pesticide facilitates the development of modern agriculture widely, for agriculture production is retrieved in a large number because of loss that the reasons such as phytopathy, worm, crop smothering cause, and drastically increase efficiency and the mechanization degree of agriculture production, in the modernization of agriculture production, play immeasurable effect.But agricultural chemicals is some toxic and harmful substances, after they enter ecotope, not only kills the biology to human hazard simultaneously, also serious harm is caused to the ecosystem simultaneously.A large amount of uses of agricultural chemicals cause Residual Pesticides in Farm Produce to exceed standard, and have had a strong impact on the safety in production of the healthy of people and agricultural-food.Along with the raising of people's living standard and the enhancing of environmental protection consciousness, the direct harm that the whole society brings pesticide residue and potential impact are also more and more paid close attention to, the clean green agricultural product that people pollute in the urgent need to non agricultural chemical residuum.But from agricultural form and the demographic situation of current China, it is impracticable for reducing to stop Pesticide use the production model that farm output is cost, and continuation is also played huge effect as the effective means of control phytopathy, worm, crop smothering by agricultural chemicals in agriculture production.This double-barreled question how solved in agriculture production just becomes study hotspot.Laboratory and real application research all show to utilize microorganism to the pesticide residue of degrading in soil, thus reduce the pesticide residue in the agricultural-food such as grain, vegetables, tealeaves, and the quality and the economic worth that improve agricultural-food are feasible.
Summary of the invention
The object of the present invention is to provide a kind of piperazine humulone degradation bacteria (Rhodococcussp.) QCT10.
Another object of the present invention is to the application of above-mentioned piperazine humulone degradation bacteria QCT10 in degraded piperazine humulone and/or production piperazine humulone bacterium for degradating residual agricultural chemical.
The present invention also aims to for the practical problems in production practice and demand, develop a kind of novel piperazine humulone bacterium for degradating residual agricultural chemical.
A further object of the present invention is the application of above-mentioned microbial inoculum in degraded piperazine humulone.
Object of the present invention realizes by following technical scheme:
The invention provides a kind of piperazine humulone degradation bacteria QCT10, for gram-positive microorganism, through being accredited as rhodococcus (Rhodococcussp.), on June 29th, 2015 is preserved in China typical culture collection center, and culture presevation number is: CCTCCNO:M2015392.Bacterial strain QCT10 is pink on LB solid plate, colony edge is smooth, and centre is slightly protruding.Bacterial strain QCT10 atrichia, size is 1.1-1.2 μm × tyrothricin (Fig. 1) of 1.6-1.7 μm, catalase is positive, can resist PXB.The application of piperazine humulone degradation bacteria QCT10 of the present invention in degraded piperazine humulone.
Piperazine humulone degradation bacteria QCT10 of the present invention is producing the application in piperazine humulone bacterium for degradating residual agricultural chemical.
Piperazine humulone bacterium for degradating residual agricultural chemical, prepares preferably by following methods:
1) be inoculated in by above-mentioned piperazine humulone degradation bacteria QCT10 in broth culture (extractum carnis 10 grams, peptone 5 grams, NaCl5 gram, 1000 milliliters, water, pH7.0-7.2) shaking flask, shaking culture is to logarithmic phase;
2) above-mentioned cultured bacterial classification is inoculated into 500 liters of seeding tanks by the inoculum size of 10% (v/v) of seed tank culture base, in culturing process, the air flow of sterile air is 1:0.8-1.2, stirring velocity is 180-240 rev/min, culture temperature is 30-32 DEG C, be cultured to logarithmic phase, seeding tank culture medium prescription used is: glucose 1.0wt%, NH
4nO
31.0wt%, K
2hPO
40.2wt%, MgSO
40.05wt%, NaCl0.01wt%, CaCO
30.3wt%, yeast extract paste 0.02wt%, pH value 7.2-7.5;
3) above-mentioned seed liquor is produced tank by 10% (v/v) inoculum size access of producing tank substratum to cultivate, produce tank used medium identical with seed tank culture base, in culturing process, the air flow of sterile air is 1:0.8-1.2, stirring velocity is 180-240 rev/min, culture temperature is 30-32 DEG C, whole process incubation time is 36-48 hour, after fermentation ends, thalline quantity reaches 1,000,000,000/more than ml, and the rear nutrient solution that fermented goes out tank and is directly distributed into liquid dosage form by plastic barrel or packing bottle or adopts adsorption by peat packing bag to be distributed into solid dosage.
Beneficial effect
It is low that the bacterium for degradating residual agricultural chemical using this invention to produce has production and application cost, easy to use, the advantage that removal effect is good, is adapted at national grain oil & vegetable production export base or has the regional spread of green food brand mark to use.The present invention is for preserving the ecological environment, and the protection people's is healthy, and the added value improving agricultural-food has great importance.Degradation bacteria QCT10 can, by piperazine humulone chemical residual degradation more than 50% in soil, use this degradation bacterial agent in the normal growth process of farm crop, normally to use chemical pesticide carry out prevention and control of weeds and ensure that Residual Pesticides in Farm Produce content meets green food requirement.
Present invention successfully solves piperazine humulone pesticide residue excessive problem in agriculture production, both given full play to chemical pesticide efficiently acting on fast in prevention and control of weeds, and non-toxic and non-pollution green agricultural product can have been produced again.
Accompanying drawing explanation
The transmission electron microscope of Fig. 1 degradation bacteria strains QCT10 shines
Biomaterial preservation information
A kind of piperazine humulone degradation bacteria QCT10, Classification And Nomenclature is Rhodococcussp.QCT10, is preserved in China typical culture collection center, and preservation date is on June 29th, 2015, preservation address is Wuhan, China Wuhan University, and culture presevation number is: CCTCCNO:M2015392.
Embodiment
Embodiment 1: the separation of bacterial strain, qualification
Gather long-term by piperazine humulone contaminated soil, get 15g pedotheque and be placed in the inorganic salt liquid substratum (NH that 100mL adds 50mg/L piperazine humulone
4nO
31.0 grams, K
2hPO
41.5 grams, KH
2pO
40.5 gram, MgSO
40.2 gram, NaCl1.0 gram, 1000 milliliters, water, pH7.0-7.2) in, 30 DEG C, 150r/min cultivates 7 days, the inoculum size with 7.5% is transferred to the inorganic salt liquid substratum of identical interpolation 50mg/L piperazine humulone, continuously switching 4 times.By liquid chromatography for measuring degradation effect.Get the pregnant solution that 0.5mL is effective, gradient dilution, the dilution pregnant solution of each gradient is got on the inorganic salt solid medium flat board that 100 μ L are coated in containing 50mg/L piperazine humulone agricultural chemicals, cultivates 5d for 30 DEG C.Single colony inoculation that picking flat board grows is in LB liquid medium (peptone 1 gram, yeast extract paste 0.5 gram, NaCl0.5 gram, agar powder 20 grams, 1000 milliliters, water, pH7.0-7.2) in test tube, 30 DEG C, 160r/min cultivates 1 day, get 1mLLB nutrient solution, be seeded to and be added with 50mgL
-1in the inorganic salt liquid substratum of piperazine humulone, 30 DEG C, 150r/min cultivates 5d, liquid chromatographic detection degradation effect.The bacterial strain of degradation effect is had to be degradation bacteria strains to piperazine humulone.1 strain degradation bacteria strains is obtained, called after QCT10, through being accredited as rhodococcus (Rhodococcussp.) by checking.Bacterial strain QCT10 is pink on LB solid plate, colony edge is smooth, and centre is slightly protruding.Bacterial strain QCT10 atrichia, size is 1.1-1.2 μm × tyrothricin of 1.6-1.7 μm, catalase is positive, can resist PXB.
Bacterial strain QCT10 is preserved in China typical culture collection center on June 29th, 2015, and culture presevation number is: CCTCCNO:M2015392.
Embodiment 2 bacterial strain is to the degradation effect of piperazine humulone
In inorganic salt liquid substratum, degradation bacteria QCT10 measures piperazine humulone degradation characteristic: the mono-bacterium colony of picking QCT10 is in 50mlLB liquid nutrient medium, and 30 DEG C, 160r/min shaking culture 12h, obtain fresh bacterium liquid.By cultured for 3ml fresh bacterium liquid through the centrifugal 5min of 6000r/min, abandoning supernatant, it is resuspended to add 3ml sterilized water, is the bacterium liquid after centrifugal suspension.At GMSM liquid nutrient medium (glucose 3.0 gram, the K of 20ml containing 50mg/l piperazine humulone
2hPO
41.5 grams, KH
2pO
40.5 gram, NaCl1.0 gram, MgSO
47H
2o0.2 gram, 1000 milliliters, water, pH7.0-7.2) in, the bacterium liquid after the centrifugal suspension of access 1.5ml, 30 DEG C, 150r/min cultivates, the content of the 7th day sampling and measuring piperazine humulone.The results are shown in Table 1, the degradation rate of degradation bacteria QCT10 to piperazine humulone reaches 75.3%.
Degraded in soil bacterium QCT10 measures piperazine humulone degradation characteristic: take 600g soil, add piperazine humulone, and making to contain piperazine humulone concentration in soil is 15mg/kg, adds the fresh bacterium liquid of QCT10, and mixing, makes the QCT10 concentration in soil be 10
11individual cell/gram soil, be placed in constant temperature culture under 30 DEG C of incubator dark conditions, the residual quantity of the 30th day sampling and measuring piperazine humulone in soil, the results are shown in Table 1, and degraded in soil bacterium QCT10 piperazine humulone of degrading reaches 56.1%.
The degradation effect of table 1 bacterial strain QCT10 to piperazine humulone in GMSM liquid nutrient medium sweetening of the soil
The preparation of embodiment 3 microbial inoculum and the degradation effect to piperazine humulone
The original seed of pesticide residual degrading bacteria QCT10 of the present invention is activated on LB test tube slant.By test tube slant strain inoculation in the 1000ml shaking flask containing 200ml broth culture (extractum carnis 10 grams, peptone 5 grams, NaCl5 gram, 1000 milliliters, water, pH7.0-7.2), 30 DEG C of constant-temperature shaking culture, to logarithmic phase, prepare inoculation seeding tank.Seeding tank 500 liters, charging capacity 400 liters, culture medium prescription (mass percent): glucose 1.0wt%, NH
4nO
31.0wt%, K
2hPO
40.2wt%, MgSO
40.05wt%, NaCl0.01wt%, CaCO
30.3%, yeast extract paste 0.02%, pH value 7.2-7.5.121 DEG C of high pressure moist heat sterilizations after feeding intake, after sterilizing completes and is cooled to 33 DEG C, by above-mentioned cultured shaking flask bacterial classification by 10% (volume percent, inoculum size down together) is inoculated into 500 liters of seeding tanks, be cultured to logarithmic phase, seeding tank temperature controls at 30-32 DEG C, and stirring velocity is 220 revs/min, and sterile air intake is 1:0.8.Produce tank used medium composition identical with seed tank culture base.Produce tankage 5 tons, charging capacity 4.5 tons.Production tank 1.1kg/cm after feeding intake
2pressure under, 121 DEG C of high pressure moist heat sterilizations, after being cooled to 33 DEG C after sterilizing, logical sterile air keeps sterile state for subsequent use.Tank cultivation is produced by arriving the inoculum size access of the seed liquor in the aforementioned seeding tank of logarithmic phase by 10%, postvaccinal production tank temperature controls at 30-32 DEG C, the air flow producing sterile air in the culturing process of tank is 1:0.8, stirring velocity is 220 revs/min, and whole technical process incubation time is 48 hours.After fermentation ends, thalline quantity reaches 1,000,000,000/more than ml.The rear nutrient solution that fermented goes out tank and is directly distributed into liquid dosage form by plastic barrel or packing bottle or adopts adsorption by peat packing bag to be distributed into solid fungicide formulation.
In soil, QCT10 liquid degradation microbial inoculum measures piperazine humulone degradation effect: the agricultural land soil getting 5000g life-time service piperazine humulone, piperazine humulone residual concentration is 4.7mg/kg, add QCT10 liquid degradation microbial inoculum prepared by the present embodiment, mixing, makes the QCT10 concentration in soil be 10
8individual cell/gram soil, is placed in 30 DEG C, and timing sampling measures the residual quantity of piperazine humulone in soil, and the results are shown in Table 2, in soil, QCT10 liquid degradation microbial inoculum reaches 52.3% to piperazine humulone degradation rate in soil.
Show 2QCT10 piperazine humulone degradation bacterial agent in soil to the degradation effect of piperazine humulone
Substratum | Soil |
Degradation rate | 52.3% |
Claims (7)
1. piperazine humulone degradation bacteria (Rhodococcussp.) QCT10, on June 29th, 2015 is preserved in China typical culture collection center, and culture presevation number is: CCTCCNO:M2015392.
2. the application of piperazine humulone degradation bacteria QCT10 according to claim 1 in degraded piperazine humulone.
3. piperazine humulone degradation bacteria QCT10 according to claim 1 is producing the application in piperazine humulone bacterium for degradating residual agricultural chemical.
4. the microbial inoculum prepared by piperazine humulone degradation bacteria QCT10 according to claim 1.
5. microbial inoculum according to claim 4, is characterized in that being prepared from by the following method:
1) be inoculated in broth culture shaking flask by piperazine humulone degradation bacteria QCT10 according to claim 1, shaking culture is to logarithmic phase;
2) above-mentioned cultured bacterial classification is inoculated into 500 liters of seeding tanks by the inoculum size of 10% (v/v) of seed tank culture base, in culturing process, the air flow of sterile air is 1:0.8-1.2, stirring velocity is 180-240 rev/min, culture temperature is 30-32 DEG C, be cultured to logarithmic phase, seeding tank culture medium prescription used is: glucose 1.0wt%, NH
4nO
31.0wt%, K
2hPO
40.2wt%, MgSO
40.05wt%, NaCl0.01wt%, CaCO
30.3wt%, yeast extract paste 0.02wt%, pH value 7.2-7.5;
3) above-mentioned seed liquor is produced tank by 10% (v/v) inoculum size access of producing tank substratum to cultivate, produce tank used medium identical with above-mentioned seed tank culture base, in culturing process, the air flow of sterile air is 1:0.8-1.2, stirring velocity is 180-240 rev/min, culture temperature is 30-32 DEG C, whole process incubation time is 36-48 hour, after fermentation ends, thalline quantity reaches 1,000,000,000/more than ml, and the rear nutrient solution that fermented goes out tank and is directly distributed into liquid dosage form by plastic barrel or packing bottle or adopts adsorption by peat packing bag to be distributed into solid dosage.
6. the application of the microbial inoculum described in claim 4 or 5 in degraded piperazine humulone.
7. application according to claim 6, is characterized in that the application of described microbial inoculum piperazine humulone pesticide residue in degraded soil.
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107699523A (en) * | 2017-11-03 | 2018-02-16 | 南京农业大学 | One plant of can degrade insecticide Buprofezin and the bacterium of Biphenthrin and its microbial inoculum of production |
CN108130288A (en) * | 2017-12-15 | 2018-06-08 | 浙江工业大学 | The application of Rhodococcus ruber and its degradable organic pollutant |
CN111979128A (en) * | 2020-09-04 | 2020-11-24 | 广西壮族自治区亚热带作物研究所(广西亚热带农产品加工研究所) | Screening method of plant-derived pesticide efficient degrading bacteria |
CN112920971A (en) * | 2021-03-17 | 2021-06-08 | 青海省农林科学院 | Fluorochloridone degrading strain and application thereof |
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107699523A (en) * | 2017-11-03 | 2018-02-16 | 南京农业大学 | One plant of can degrade insecticide Buprofezin and the bacterium of Biphenthrin and its microbial inoculum of production |
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CN111394274B (en) * | 2017-11-03 | 2021-10-22 | 南京农业大学 | Microbial inoculum for degrading pesticides buprofezin and bifenthrin and application thereof |
CN108130288A (en) * | 2017-12-15 | 2018-06-08 | 浙江工业大学 | The application of Rhodococcus ruber and its degradable organic pollutant |
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CN111979128A (en) * | 2020-09-04 | 2020-11-24 | 广西壮族自治区亚热带作物研究所(广西亚热带农产品加工研究所) | Screening method of plant-derived pesticide efficient degrading bacteria |
CN112920971A (en) * | 2021-03-17 | 2021-06-08 | 青海省农林科学院 | Fluorochloridone degrading strain and application thereof |
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