CN101157901A - Chlorpyrifos pesticide degradation strain, inocula and preparation method thereof - Google Patents
Chlorpyrifos pesticide degradation strain, inocula and preparation method thereof Download PDFInfo
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- CN101157901A CN101157901A CNA2007100303042A CN200710030304A CN101157901A CN 101157901 A CN101157901 A CN 101157901A CN A2007100303042 A CNA2007100303042 A CN A2007100303042A CN 200710030304 A CN200710030304 A CN 200710030304A CN 101157901 A CN101157901 A CN 101157901A
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- degradation
- chlopyrifos
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- pesticide
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Abstract
The invention discloses a chlorpyrifos pesticide-degrading bacterium which refers to a Hu-01strain pertaining to Cladosporium sp. The preservation number of the strain is CCTCC M207111, and a Genebank accession number of the ITS sequence analysis is EF405864. Meantime, the invention discloses a method of preparation of inocula. The invention has the advantages of low production cost of inocula, convenient operation and good degradation effect. The invention is suitable for being widely used in fruit and vegetable production and export bases in China or in places possessing green food trademark, which has great significances in promoting the production of uncontaminated vegetable and green food.
Description
Technical field
The present invention relates to microbial technology field, especially relate to a kind of chlopyrifos pesticide degradation bacteria, its microbial inoculum and preparation method.
Technical background
Fruit, vegetables are the requisite daily necessities of people, and agricultural chemicals is insect pests crop smothering in the fruits and vegetables production process, the requisite selection of assurance output and quality.
Guarantee that garden stuff pesticide residue meets food hygienic standard, relate to every citizen's life and health, become the important content that governments at all levels implement " vegetable basket, fruit basket " engineering.Simultaneously, along with improving constantly of standard of living, " green food, nuisanceless fruits and vegetables " go deep into the popular feelings day by day, and garden stuff pesticide residue also becomes one of focus of public attention.
Under various factors comprehensive actions such as interests driving, the harm of sick worm, user's quality, the not science service condition of agricultural chemicals is generally serious, and directly caused the severe overweight of garden stuff pesticide residue, threaten people's life security and health, and to having influenced China's fruits and vegetables foreign exchange earning to a great extent.The world market is maximum direct point of penetration with pesticide residue, and China's outlet fruits and vegetables are set up strict " green barrier ".Departments such as inspection and quarantining for import/export general bureau of country, the Ministry of Agriculture point out that pesticide residue have become main hindering factor and " bottleneck " of China's fruits and vegetables foreign exchange earning, must pay much attention to the source control of garden stuff pesticide residue and administer new industrial research.
It is the crucial supporting technology of food safety production that garden stuff pesticide residue is administered new technology.Control pesticidal contamination source and residual improvement new industrial research are the basic ways that solves pesticide residue in the food.The research that reduces pesticide residual contamination at present concentrates on mostly selects high-efficiency low-toxicity low residue chemical pesticide and biological pesticide, improvement formulation and utilisation technology aspect for use, but produces little effect.The use of agricultural chemicals is indispensable in the agricultural-food production process, seek efficient, safe, economic agricultural-food pesticide residue and handle new technology, solve the contradiction of prevention and control of plant diseases, pest control medication and pesticide residue, become the great scientific research proposition that researcher needs to be resolved hurrily with great economy and social effect.Utilizing the microbiological deterioration agent to handle agricultural-food, is frontier, new way that pesticide residue control is handled, and many developed countries and enterprise of major company drop into huge fund to be engaged in the research and development of pesticide residue efficient degrading bacteria.Laboratory and real application research all show and utilize degrade pesticide residue in the fruits and vegetables of microorganism, can effectively improve the quality and the economic worth of agricultural-food.
Chlorpyrifos 94 is to use a kind of very widely agricultural chemicals in recent years, does not also have to find special degradation bacterial agent at chlopyrifos pesticides at present.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of new chlopyrifos pesticide degradation bacteria is provided.
Another purpose of the present invention has provided the method that realizes described chlopyrifos pesticide degradation bacteria.
Of the present invention also have a purpose to provide the method for using described chlopyrifos pesticide degradation bacteria to prepare microbial inoculum, the microbial inoculum that obtains can make the residual quantity of organophosphorus pesticides such as Chlorpyrifos 94 reduce more than 85%, have higher degradation effect, production and use cost are lower.
Technical scheme of the present invention provides a kind of chlopyrifos pesticide degradation bacteria, it is characterized in that described degradation bacteria is the Hu-01 bacterial strain that cladosporium belongs to Cladosporium sp., culture presevation number is CCTCC M 207111, and the Genebank number of landing of this bacterial strain ITS sequential analysis is EF405864.
The preparation method of described chlopyrifos pesticide degradation bacteria may further comprise the steps:
Bacterial classification adopts sand pipe preservation method to preserve.The bacterial classification of chlopyrifos pesticide degradation bacteria Hu-01 is activated on culture dish, be inoculated in the test tube slant.Look into Bi Shi substratum (Czapek medium) according to following formulated: SODIUMNITRATE: 2.0g, Repone K: 0.5g, sal epsom: 0.5g, dipotassium hydrogen phosphate: 1.0g, ferric sulfate: 0.01g, sucrose: 30.0g, distilled water: 1000mL.Look in the Bi Shi substratum at the liquid of pH7.0, insert the 0.1g wet thallus,, cultivate 1-7d in the shaking table of 150r/min at 28 ℃.
The invention provides the method that adopts described chlopyrifos pesticide degradation bacteria to prepare degradation bacterial agent, may further comprise the steps:
(1) bacterial classification with chlopyrifos pesticide degradation bacteria Hu-01 activates on culture dish, is inoculated in the test tube slant;
(2) the inclined-plane kind of chlopyrifos pesticide degradation bacteria Hu-01 is inoculated in looks in the Bi Shi culture media shaking vase, shaking culture is to logarithmic phase;
(3) (2) bacterial classification is gone into seeding tank according to 8%～10% inoculum size kind, be cultured to logarithmic phase;
(4) will arrive the logarithmic phase seed liquor inoculates into the production jar according to 8%～10% inoculum size and cultivates;
(5) nutrient solution gets bacterium liquid through the centrifuging and taking supernatant, and bacterium liquid adds protective material immediately, is distributed into liquid dosage form.
The air flow that the described seed tank culture condition of step (3) is a sterile air is 45～60L/min, and stirring velocity is 200～300 rev/mins, 28 ℃ of temperature.
The used culture medium prescription of step (3) seeding tank is NaNO
30.2%, K
2HPO
40.1%, KCl 0.05%, MgSO
40.05%, Fe
2(SO
4)
30.001%, sucrose 3%, peptone 0.5%.
The described seeding tank of step (3) feeds intake by volumetric quantity 80%, feed intake finish after at 1.1kg/cm
3Pressure, 121 ℃ of conditions under sterilize, be cooled to 28 ℃ of reserve inoculations.
The air flow that the described culture condition of producing jar of step (4) is a sterile air is 0.45～0.60m
3/ min, stirring velocity is 100～200 rev/mins, and culture temperature is 26～30 ℃, and incubation time is 88～96 hours.
The described production of step (a 4) jar used culture medium prescription is NaNO
30.2%, K
2HPO
40.1%, KCl 0.05%, MgSO
40.05%, Fe
2(SO
4)
30.001%, sucrose 3%, peptone 0.5%.
The described production of step (4) jar is according to charging capacity: capacity=0.08～0.1: 1 ratio feeds intake, and the back that feeds intake is at 1.1kg/cm
3Pressure, 121 ℃ of conditions under sterilize, be cooled to 28 ℃ after, logical sterile air keeps sterile state to be equipped with inoculation.
It is 0.7% that the described protective material of step (5) consists of the sodium-chlor massfraction, and the glycine massfraction is 0.35%, and the Sodium Benzoate massfraction is 0.03%, and triumphant loose volume fraction is 0.17076%, and the volume fraction of glycerine is 8.37801%.
The invention provides a kind of chlopyrifos pesticide degradation bacteria Hu-01, be deposited at Chinese typical culture collection center (CCTCC) on July 20th, 2007, culture presevation number is CCTCCM207111, belongs to Cladosporium sp. through being accredited as cladosporium.Main biological characteristics is: bacterial strain is dark culturing on PDA, bacterium colony olive green, reverse side blackish green.Conidiophore is branch not, and is upright.0～1 barrier film of branch spore, (5～27) * (2～2.5) μ m.Give birth to or adnation on conidium top, and oval, lemon shape is to subsphaeroidal, light olive look, no barrier film, one or both ends tool spore navel, (4～8) * (2～3) μ m.The Genebank number of landing of this bacterial strain ITS sequential analysis is EF405864.
The processing step that uses described chlorpyrifos pesticide residue efficient degrading bacteria to produce microbial inoculum is: inclined-plane kind-shake bottle kind cultivation one seed tank culture-production jar to cultivate-be packaged as liquid bacterial agent.
The invention has the beneficial effects as follows:
(1) efficient degradation fungi Hu-01 can make the residual quantity of Chlorpyrifos 94 reduce more than 85%, use this degradative fungi microbial inoculum, successfully solved the contradiction of the use and efficient, safe, the economic prevention and elimination of disease and pests of agricultural chemicals in the agricultural-food production process, can produce non-toxic and non-pollution green agricultural product again, can in the normal growth process of fruits and vegetables, use the chemical pesticide control disease and pest and guarantee that the agricultural chemicals residual content meets the green food requirement in the fruits and vegetables.
(2) to have a production cost low, easy to use for the pesticide residue high efficiency degradation bacterial agent produced of the present invention, and the advantage of good degrading effect is adapted at national fruits and vegetables production export base or has the local big area of green food brand mark to use.
(3) the present invention is to promoting coordinating and unifying prevention and control of plant diseases, pest control medication and food safety, and large-scale promotion is used fruits and vegetables agricultural chemicals microbiological treatment, and production pollution-free vegetable, green food have great importance.
Embodiment
Further describe the present invention below in conjunction with specific embodiment.
The preparation method of embodiment 1 chlopyrifos pesticide degradation bacteria Hu-01 and upgrowth situation are measured
The bacterial classification of chlopyrifos pesticide degradation bacteria Hu-01 is activated on culture dish, be inoculated in the test tube slant.Look into Bi Shi substratum (Czapek medium) according to following formulated: SODIUMNITRATE: 2.0g, Repone K: 0.5g, sal epsom: 0.5g, dipotassium hydrogen phosphate: 1.0g, ferric sulfate: 0.01g, sucrose: 30.0g, distilled water: 1000mL.Look in the Bi Shi substratum at the liquid of pH7.0, insert the 0.1g wet thallus,, cultivate 1-7d in the shaking table of 150r/min at 28 ℃.
The relation of the upgrowth situation of incubation time and degradation bacteria Hu-01 sees Table 1.
Table 1 incubation time is to the influence of chlopyrifos pesticide degradation bacteria mycelium dry weight
| Mycelium dry weight (g/50mL) | ||||
| 3d | 4d | 5d | 6d | 7d |
| 0.2825±0.002 | 0.3869±0.002 | 0.4520±0.004 | 0.4458±0.002 | 0.4371±0.003 |
Annotate: above result is 3 repetition mean numbers.
Embodiment 2
(1) bacterial classification with chlopyrifos pesticide degradation fungi Hu-01 activates on culture dish, and measures degradation property, is inoculated on the test tube slant standby;
(2) the test tube kind is inoculated in and contains 250mL and look into Bi Shi substratum 1000mL and shake in the bottle, and constant-temperature shaking culture is prepared the inoculation seeding tank to logarithmic phase;
(3) seeding tank is 100 liters, 80 liters of charging capacitys, and culture medium prescription is: NaNO
30.2%, K
2HPO
40.1%, KCl 0.05%, MgSO
40.05%, Fe
2(SO
4)
30.001%, sucrose 3%, peptone 0.5%, feed intake finish after 1.1kg/cm
3Pressure under, 121 ℃ of high pressure moist heat sterilizations, be cooled to 28 ℃ after, the above-mentioned cultured bacterium bacterial classification that connects is inoculated in 100 liters of seeding tanks by 8%～10% inoculum size, be cultured to logarithmic phase, stirring velocity is 200～300 rev/mins, and the sterile air air flow is 45～60L/min.
(4) seed liquor that will arrive logarithmic phase puts into production by 8%～10% inoculum size and jar cultivates, and it is identical with the seed tank culture base to produce jar used medium component.Produce 1 ton of tankage, charging capacity is 0.08～0.1 ton.Production jar after feeding intake, 1.1kg/cm
3Pressure under, 121 ℃ of high pressure moist heat sterilizations, be cooled to 28 ℃ after, logical sterile air keeps sterile state standby.A postvaccinal production jar temperature is controlled at 28 ℃, and the sterile air air flow is 0.45～0.60m
3/ min, stirring velocity is 100～200 rev/mins, it is 88～96 hours that the whole technology of this step is cultivated the flow process time.
(5) nutrient solution centrifuging and taking supernatant under the 12000r/min condition got bacterium liquid after fermentation was finished; bacterium liquid adds following protective material immediately: the sodium-chlor massfraction is 0.7%; the glycine massfraction is 0.35%; the Sodium Benzoate massfraction is 0.03%; triumphant loose volume fraction is 0.17076%; the volume fraction of glycerine is 8.37801%, is distributed into liquid dosage form.
One, be in the liquid nutrient medium of sole carbon source Chlorpyrifos 94 with 50mg/L, inoculation 0.2% grow to logarithmic phase, preserve different time Hu-01 bacterium liquid, shaking table is cultivated the degraded situation of high performance liquid chromatography detection agricultural chemicals after 24 hours, degradation rate can reach more than 85%, the results are shown in Table 2.
Table 2 degradation bacteria Hu-01 is to the degradation property of Chlorpyrifos 94
| Shelf time (d) | 7 | 14 | 30 | 60 |
| Degradation rate (%) | 97.68 | 93.49 | 90.95 | 86.09 |
Two, carry out field pesticides residue degrading experiment and demonstration with microbial inoculum of the present invention.
Choose the cabbage heart of non-dosed agricultural chemicals in the field, mark off 18 sub-districts, every sub-district 6m
2, spraying the 48% Le Siben missible oil of 500x, 800x, 1200x respectively with portable atomizer, formulation rate is 50kg/667m
2, contrast and processing are alternately.Evenly sprayed degradation germ liquid in 3 days after the dispenser, every sub-district 0.3L compares with clear water, sprays bacterium liquid and gets the plant sample according to five point samplings after 1,2,3 days and measure its pesticide residue content, result such as table 3.The result shows that degradation bacteria liquid can effectively remove chlopyrifos residue, and good degradation effect is arranged.
The chlopyrifos residue degraded test of table 3 degradation bacteria Hu-01 field
| Time (d) | Dispenser dose (ml/667m 2) | |||
| 100 | 160 | 240 | ||
| Degradation rate (%) | 1 2 3 | 72.09± 0.52b 72.91± 1.29b 57.82± 1.50ab | 81.18± 1.20a 85.07± 1.32a 69.86± 1.89a | 78.81± 2.23a 57.37± 1.77c 54.27± 6.33b |
Annotate: same line data (mean value ± standard error) has different English alphabet persons and is illustrated in 0.05 level difference significantly (DMRT method) in the table.
Claims (10)
1. a chlopyrifos pesticide degradation bacteria is characterized in that described degradation bacteria is the Hu-01 bacterial strain that cladosporium belongs to Cladosporium sp., and culture presevation number is CCTCC M 207111, and the Genebank number of landing of this bacterial strain ITS sequential analysis is EF405864.
2. method that realizes the described chlopyrifos pesticide degradation bacteria of claim 1, it is characterized in that may further comprise the steps: bacterial classification adopts sand pipe preservation method to preserve, the bacterial classification of chlopyrifos pesticide degradation bacteria Hu-01 is activated on culture dish, be inoculated in the test tube slant, look into the Bi Shi substratum according to following formulated: SODIUMNITRATE: 2.0g, Repone K: 0.5g, sal epsom: 0.5g, dipotassium hydrogen phosphate: 1.0g, ferric sulfate: 0.01g, sucrose: 30.0g, distilled water: 1000mL looks in the Bi Shi substratum at the liquid of pH7.0, inserts the 0.1g wet thallus, at 28 ℃, cultivate 1-7d in the shaking table of 150r/min.
3. method that adopts the described chlopyrifos pesticide degradation bacteria of claim 1 to prepare degradation bacterial agent is characterized in that may further comprise the steps:
(1) bacterial classification with chlopyrifos pesticide degradation bacteria Hu-01 activates on culture dish, is inoculated in the test tube slant;
(2) the inclined-plane kind of chlopyrifos pesticide degradation bacteria Hu-01 is inoculated in looks in the Bi Shi culture media shaking vase, shaking culture is to logarithmic phase;
(3) step (2) bacterial classification is gone into seeding tank according to 8%～10% inoculum size kind, be cultured to logarithmic phase;
(4) will arrive the logarithmic phase seed liquor inoculates into the production jar according to 8%～10% inoculum size and cultivates;
(5) nutrient solution gets bacterium liquid through the centrifuging and taking supernatant, and bacterium liquid adds protective material immediately, is distributed into liquid dosage form.
4. according to the described method for preparing degradation bacterial agent of claim 3, it is characterized in that the air flow that the described seed tank culture condition of step (3) is a sterile air is 45～60L/min, stirring velocity is 200～300 rev/mins, 28 ℃ of temperature.
5. according to the described method for preparing degradation bacterial agent of claim 3, it is characterized in that the used culture medium prescription of step (3) seeding tank is NaNO
30.2%, K
2HPO
40.1%, KCl0.05%, MgSO
40.05%, Fe
2(SO
4)
30.001%, sucrose 3%, peptone 0.5%.
6. according to the described method for preparing degradation bacterial agent of claim 3, it is characterized in that the described seeding tank of step (3) feeds intake by volumetric quantity 80%, feed intake finish after at 1.1kg/cm
3Pressure, 121 ℃ of conditions under sterilize, be cooled to 28 ℃ of reserve inoculations.
7. according to the described method for preparing degradation bacterial agent of claim 3, it is characterized in that the air flow that the described culture condition of producing jar of step (4) is a sterile air is 0.45～0.60m
3/ min, stirring velocity is 100～200 rev/mins, and culture temperature is 26～30 ℃, and incubation time is 88～96 hours.
8. according to the described method for preparing degradation bacterial agent of claim 3, it is characterized in that the described production of step (a 4) jar used culture medium prescription is NaNO
30.2%, K
2HPO
40.1%, KCl 0.05%, MgSO
40.05%, Fe
2(SO
4)
30.001%, sucrose 3%, peptone 0.5%.
9. according to the described method for preparing degradation bacterial agent of claim 3, it is characterized in that the described production of step (4) jar is according to charging capacity: capacity=0.08～0.1: 1 ratio feeds intake, and the back that feeds intake is at 1.1kg/cm
3Pressure, 121 ℃ of conditions under sterilize, be cooled to 28 ℃ after, logical sterile air keeps sterile state to be equipped with inoculation.
10. according to the described method for preparing degradation bacterial agent of claim 3; it is characterized in that it is 0.7% that the described protective material of step (5) consists of the sodium-chlor massfraction; the glycine massfraction is 0.35%; the Sodium Benzoate massfraction is 0.03%; triumphant loose volume fraction is 0.17076%, and the volume fraction of glycerine is 8.37801%.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100303042A CN101157901A (en) | 2007-09-18 | 2007-09-18 | Chlorpyrifos pesticide degradation strain, inocula and preparation method thereof |
| CN2008102152592A CN101469313B (en) | 2007-09-18 | 2008-09-18 | Chlopyrifos pesticide degradation bacteria, inocula and preparation thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100303042A CN101157901A (en) | 2007-09-18 | 2007-09-18 | Chlorpyrifos pesticide degradation strain, inocula and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101157901A true CN101157901A (en) | 2008-04-09 |
Family
ID=39306140
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007100303042A Pending CN101157901A (en) | 2007-09-18 | 2007-09-18 | Chlorpyrifos pesticide degradation strain, inocula and preparation method thereof |
| CN2008102152592A Expired - Fee Related CN101469313B (en) | 2007-09-18 | 2008-09-18 | Chlopyrifos pesticide degradation bacteria, inocula and preparation thereof |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008102152592A Expired - Fee Related CN101469313B (en) | 2007-09-18 | 2008-09-18 | Chlopyrifos pesticide degradation bacteria, inocula and preparation thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN101157901A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101966530A (en) * | 2010-09-09 | 2011-02-09 | 浙江大学 | Bioremediation method of soil polluted by chlorpyrifos |
| CN102703327A (en) * | 2012-02-04 | 2012-10-03 | 西北农林科技大学 | Cladosporium Sp. XJ-AC03 and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2844922Y (en) * | 2005-12-06 | 2006-12-06 | 万积成 | Gel gold test paper for quick inspecting remained 3,5,6-trichlorodipyridine-one |
| CN100371436C (en) * | 2005-12-23 | 2008-02-27 | 南京农业大学 | Bacterium for degrading chlorpyrifos pesticide residue and produced bacterium formulation |
-
2007
- 2007-09-18 CN CNA2007100303042A patent/CN101157901A/en active Pending
-
2008
- 2008-09-18 CN CN2008102152592A patent/CN101469313B/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101966530A (en) * | 2010-09-09 | 2011-02-09 | 浙江大学 | Bioremediation method of soil polluted by chlorpyrifos |
| CN101966530B (en) * | 2010-09-09 | 2012-07-04 | 浙江大学 | Bioremediation method of soil polluted by chlorpyrifos |
| CN102703327A (en) * | 2012-02-04 | 2012-10-03 | 西北农林科技大学 | Cladosporium Sp. XJ-AC03 and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101469313A (en) | 2009-07-01 |
| CN101469313B (en) | 2011-02-16 |
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