CN101974453B - Pyrethroid pesticide degrading bacteria and bactericide thereof - Google Patents

Pyrethroid pesticide degrading bacteria and bactericide thereof Download PDF

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CN101974453B
CN101974453B CN2010102841003A CN201010284100A CN101974453B CN 101974453 B CN101974453 B CN 101974453B CN 2010102841003 A CN2010102841003 A CN 2010102841003A CN 201010284100 A CN201010284100 A CN 201010284100A CN 101974453 B CN101974453 B CN 101974453B
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钟国华
胡美英
陈少华
刘晶晶
黄文文
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South China Agricultural University
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Abstract

The invention discloses pyrethroid pesticide degrading bacteria and a bactericide thereof, belonging to the field of biological high-tech. The adopted strain ZS-S-01 is identified as Stenotrophomonas sp. with the preservation number of CCTCC M 2010095, wherein, the Genbank registry number of the 16S rDNA of the strain is HM016874. The main biological characteristics are as follows: the strain is Gram-negative and has multiple polar flagellum and cells in a short rod shape; and the bacterial colony is milky white and slight yellowish, thicker and non-transparent, the diameter is 0.5mm-1mm, the edge is regular and orderly, the middle part is slightly protruding, and the surface is smooth and bright without wrinkles. The bactericide of the invention has the advantages of low production cost, convenient use and good degradation effect, thus being applicable to the national fruit and vegetable production and export bases or places with green food brand marks at a large area. The bactericide is of great significance to promoting production of nuisance-free vegetables and green food.

Description

Pyrethroids pesticide degradation bacterium and microbial inoculum thereof
Technical field
The present invention provides a kind of pyrethroids pesticide degradation bacterium and microbial inoculum thereof, belongs to biological high-tech field, is to utilize method of microorganism degraded chemical pesticide residual, is applicable to the production of green non-polluted farm product in the modern agriculture production.
Background technology
Pyrethroid pesticide is one type of bionical sterilant by the simulation natural pyrethrum of synthetic; It is an action target with neural sodium-ion channel; Have that consumption is low, drug effect is high, insecticidal spectrum extensively reaches advantages such as toxicity is little, be widely used in the control of various pests such as cotton, tealeaves, paddy rice, fruit tree, vegetables and domestic hygiene insect.Especially in the last few years; Because the poisoning of severe toxicity such as organophosphorus class agricultural chemicals and because of pesticide residue " green barrier " event frequency of meeting with life that takes place frequently that exceeds standard causes the great attention of countries in the world, makes relevant regulations one after another; The use of the high poison of strict restriction, highly toxic pesticide; Simultaneously development of new, efficient, low toxicity substitute products energetically so that pyrethrin is able to is flourish, become one of main agricultural chemicals type of domestic replacement organochlorine pesticide and other hypertoxic long residual sterilant.At present its kind number and usage quantity are only second to organophosphorus pesticide, occupy second of sterilant market, account for 1/4 of agricultural chemicals market, the world, have accounted for insecticidal total at the usable floor area of China and have used more than 1/3 of area.Absatzvolumen maximum kind in the pyrethrin whole world has PP-383, fenvalerate, Deltamethrin and FCR-1272 etc., and their sales volume reaches respectively more than 100,000,000 dollars every year.Frequent use along with pyrethrin; Particularly the later pyrethrin of the s-generation has light, heat-staple characteristics; Transformation period is longer in environment; Be difficult to degraded fast under field conditions (factors), thereby the pyrethrin residue problem is serious day by day, has become one of 3 main in China outlet vegetables, fruit pesticide residues at present.European Union rose in the pesticide residue new standard of enforcement on July 1st, 2000, and pyrethrin maximum residue limit standard change is maximum, and standard has improved tens to hundreds of times, the part agricultural byproducts outlet of China was brought seriously influence.Along with people's growth in the living standard in recent years, people press for the cleaning green food that non agricultural chemical residuum pollutes.
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 skills that solves pesticide residue in the food.The research that reduces pesticide residual contamination at present concentrates on chemical pesticide and biological pesticide, improvement formulation and the utilisation technology aspect of selecting the high-efficiency low-toxicity low residue for use mostly, but produces little effect.Agricultural chemicals can not reduce 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.Utilize the microbiological deterioration agent to handle agricultural-food; Be frontier, the new way that pesticide residue control is handled; Have advantages such as easy and simple to handle, economical and practical, non-secondary pollution, present many developed countries and enterprise of major company all 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 the pesticide residue of utilizing in the microbiological deterioration fruits and vegetables, and the quality and the economic worth that improve agricultural-food are feasible.This seminar has had the patent of invention (200710030304.2, publication number: CN 101157901A, open day on April 9th, 2008) of degradation bacteria in the prior art, and this patent is that a strain cladosporium belongs to Hu-01, effectively the chlorpyrifos degradation pesticide residue.
Fenvalerate is to use a kind of very widely pyrethroid pesticide in recent years, does not also have to find special degradation bacterium preparation to the fenvalerate pesticide residue at present.
Summary of the invention
The object of the invention provides a kind of new pyrethroids pesticide degradation bacterium, and develops out a kind of novel pesticide residual degrading bacteria microbial inoculum in the deficiency to prior art.Use said pyrethroids pesticide degradation bacterium can make the residual quantity of pyrethroid pesticides such as fenvalerate, Deltamethrin, beta_cypermethrin, betacyfluthrin reduce more than 80%; Have higher degradation effect, production and use cost are low.
Invention realizes above-mentioned purpose through following technical scheme:
The present invention provides a kind of pyrethroids pesticide degradation bacterium; It is characterized in that said degradation bacteria is ZS-S-01; Through being accredited as oligotrophy zygosaccharomyces (Stenotrophomonas sp.); Be deposited at Chinese typical culture collection center (CCTCC) (Wuhan University preservation center) on April 21st, 2010, culture presevation number is CCTCC M 2010095.Main biological characteristics is a Gram-negative, extremely gives birth to many flagellums, the cell rod-short, and size is 0.5 μ m～0.6 μ m * 0.4 μ m～0.5 μ m; Bacterium colony is milky white yellow partially, diameter 0.5mm～1mm, and regular edges is neat, and the centre has projection slightly, and bacterium colony is thicker, and is opaque, and smooth surface is shinny, not shrinkage.Citrate test, acetyl methyl carbinol (V-P) test, the gelatin liquification test positive; Nitrate reduction test, the test of triple sugariron deep agar, cellulose hydrolysis test, starch hydrolysis experiment, methyl red (MR) are tested, milk solidifies with peptonize test, hydrogen sulfide produces the test feminine gender; Can utilize sucrose, fructose, semi-lactosi, wood sugar, glucose and seminose, the Genbank accession number of the 16S rDNA of this bacterial strain is HM016874.
This bacterium is to adopt the enrichment culture method screening to obtain: the basic medium of 100mL is installed in the 250mL triangular flask sterilize, cooling back aseptic condition adds pyrethrin pesticide down, and to make its final concentration be 100mgL -1, the mud soil sample 10g that is incorporated in Guangdong Province's collection simultaneously is in 30 ℃, 180rmin -1After shaking table was cultivated 7d, the inoculum size by 10% was transferred to second batch and contains 250mgL -1In the basic medium of pyrethrin pesticide.After the same terms is cultivated 7d, be transferred to by 10% inoculum size again and contain pyrethrin pesticide and be 500mgL -1Basic medium in, continue to cultivate 7d.Get 0.1mL basic medium fermented liquid this moment and carry out the plate streaking separation repeatedly,, single colony inoculation to the test tube slant substratum, is preserved in 4 ℃ of refrigerators until obtaining single bacterium colony.Basic medium wherein is: NH 4NO 31.0g, MgSO 47H 2O 0.5g, (NH 4) 2SO 40.5g, KH 2PO 40.5g, NaCl0.5g, K 2HPO 41.5g, H 2O 1000mL, pH 7.0.
The technology of using said fenvalerate pesticide residual degrading bacteria to produce microbial inoculum is: inclined-plane kind-shake bottle kind-seed tank culture-production jar to cultivate-be packaged as liquid dosage form.Detailed implementation step of the present invention is:
(1) with the bacterial classification inoculation of fenvalerate pesticide residual degrading bacteria ZS-S-01 in looking into the Bi Shi culture media shaking vase, shaking culture is to logarithmic phase;
(2) above-mentioned cultured bacterial classification is inoculated into seeding tank by volume ratio 8%～10% inoculum size of seed tank culture base, be cultured to logarithmic phase, the used mass percent culture medium prescription of seeding tank is: sucrose 3%, NaNO 30.2%, KCl 0.05%, MgSO 40.05%, K 2HPO 40.1%, Fe 2(SO 4) 30.001%, peptone 0.05%, pH=7.0;
The seed liquor that (3) will reach logarithmic phase is inoculated into the cultivation of production jar according to 8%～10% inoculum size, and it is identical with the seed tank culture base to produce jar used substratum;
The air flow of sterile air is 0.45～0.60m in the culturing process of seeding tank and production jar 3/ min, stirring velocity is 200～300rmin -1, 30 ℃ of temperature, the whole process incubation time is 36～48h, the nutrient solution that obtains is microbial inoculum.
Pyrethroids pesticide degradation bacterium of the present invention can be used for degradation of pyrethroid pesticide residue, as be used to degrade fenvalerate, Deltamethrin, beta_cypermethrin or betacyfluthrin etc.It is 1.0 * 10 that 50mL concentration is used in every square metre of farmland of application quantity 7CFUmL -1Degradation germ liquid.
Compared with prior art, the present invention has following beneficial effect:
(1) pyrethroids pesticide degradation bacterium ZS-S-01 can make the residual quantity of pyrethroid pesticides such as fenvalerate, Deltamethrin, beta_cypermethrin, betacyfluthrin reduce more than 80%; Use this degradation bacterium preparation; 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, use the chemical pesticide control disease and pest in the normal growth process of fruits and vegetables again and guarantee that pesticide residue 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 logo to use.
(3) the present invention promotes medication of the coordinating and unifying prevention and control of plant diseases, pest control 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.
Pyrethroids pesticide degradation bacterium ZS-S-01 of the present invention; Through being accredited as oligotrophy zygosaccharomyces (Stenotrophomonas sp.); Be deposited at Chinese typical culture collection center (CCTCC) on April 21st, 2010, culture presevation number is CCTCC M 2010095.
Embodiment
With bacterial classification activation on solid plate of pyrethroids pesticide degradation bacterium ZS-S-01 of the present invention, and measure degradation property, be inoculated on the test tube slant subsequent use.The test tube kind of fenvalerate pesticide residual degrading bacteria ZS-S-01 is inoculated in the Bi Shi substratum 1000mL that looks into that contains 250mL shakes in the bottle, constant-temperature shaking is prepared the inoculation seeding tank to logarithmic phase.Seeding tank 100L, charging capacity 80L, culture medium prescription is: sucrose 3%, NaNO 30.2%, KCl 0.05%, MgSO 40.05%, K 2HPO 40.1%, Fe 2(SO 4) 30.001%, peptone 0.05%, pH=7.0.Feed intake accomplish after at 1.1Kg/cm 3Pressure under, high pressure moist heat sterilization under 121 ℃ of conditions, be cooled to 30 ℃ after, the above-mentioned cultured bottle bacterial classification that shakes is inoculated in the 100L seeding tank by 8%～10% inoculum size, the air flow of sterile air is 0.45～0.60m 3/ min, stirring velocity is 200～300rmin -1, it is subsequent use to be cultured to logarithmic phase.The seed liquor that arrives logarithmic phase put into production according to 8%～10% inoculum size jar cultivate, 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 is at 1.1Kg/cm 3Pressure under, high pressure moist heat sterilization under 121 ℃ of conditions, be cooled to 30 ℃ after, logical sterile air, air flow is 0.45～0.60m 3/ min, stirring velocity is 200～300rmin -1, culture temperature is controlled at 30～35 ℃, and it is 36～48h that whole technology is cultivated the flow process time.
The nutrient solution that obtains directly is distributed into liquid dosage form with plastic barrel or packing bottle.
1. (component is: NH at basic salt culture medium 4NO 31.0g, NaCl 0.5g, K 2HPO 41.5g, KH 2PO 40.5g, MgSO 47H 2O 0.5g, water 1000mL, adding final concentration respectively in pH7.0) is 50mgL -1Pyrethroid pesticide such as fenvalerate, Deltamethrin, beta_cypermethrin, betacyfluthrin; Inoculation degradation bacteria ZS-S-01; And establish the substratum that do not connect bacterium and be contrast, shaking table is cultivated the degraded situation that HPLC behind the 4d detects agricultural chemicals, and the result sees table 1.It is pyrethroid pesticide remained that the result shows that degradation bacteria can effectively be removed fenvalerate, Deltamethrin, beta_cypermethrin, betacyfluthrin etc., has good degradation effect.
Table 1 degradation bacteria ZS-S-01 is to the degradation effect of fenvalerate, Deltamethrin, beta_cypermethrin, betacyfluthrin
Figure BSA00000272994300061
Annotate: above result repeats MV (
Figure BSA00000272994300062
± standard error SE) 3 times.
Degradation rate (%)=(1-C 1/ C 0) * 100%
C1: degradation bacteria is handled pesticide residue concentration (mgL -1); C 0: control treatment pesticide residue concentration (mgL -1).
2. carry out field pesticides residue degrading experiment and demonstration with microbial inoculum of the present invention.
Choose cabbage heart in the field, mark 18 sub-districts, every sub-district 6m without dispenser 2, spraying 20% the fenvalerate missible oil of 1000X, 2000X, 3000X respectively with portable atomizer, formulation rate is 2g/667m 2, contrast and processing are alternately.Evenly sprayed degradation germ liquid in 2 days after the dispenser, every sub-district 300mL does contrast with clear water, sprays bacterium liquid and gets cabbage heart appearance gas chromatography determination pesticide residue by five point samplings after 1,2,3 days, and the result sees table 2.The result shows that degradation bacterium preparation can effectively remove the beta_cypermethrin pesticide residue, has good degradation effect.
The fenvalerate degraded test of table 2 degradation bacteria ZS-S-01 field
Figure BSA00000272994300063
Annotate: 1) above result repeats MV (
Figure BSA00000272994300064
± standard error SE) 3 times.
2) be illustrated in 5% level difference not remarkable (DMRT method) with indicating same letter person behind the column data in the table.Calculation formula is following:
Figure BSA00000272994300071
In the formula: C=standard substance concentration (mgL -1)
S 1=standard substance peak area
S 2=sample peak area
V 1=standard substance sampling volume (μ L)
V 2=sample feeding volume (μ L)
G=sample quality (g)
V=constant volume (mL).

Claims (4)

1. pyrethroids pesticide degradation bacterium ZS-S-01; Be Gram-negative bacteria; Through being accredited as oligotrophy zygosaccharomyces (Stenotrophomonas sp.); Be deposited at Chinese typical culture collection center (CCTCC) on April 21st, 2010, culture presevation number is CCTCC M 2010095, and the Genbank accession number of the 16S rDNA of this bacterial strain is HM016874.
2. pyrethroids pesticide degradation bacterium microbial inoculum is characterized in that producing through following method:
(1) with the bacterial classification inoculation of fenvalerate pesticide residual degrading bacteria ZS-S-01 in looking into the Bi Shi culture media shaking vase, shaking culture is to logarithmic phase;
(2) above-mentioned cultured bacterial classification is inoculated into seeding tank by volume ratio 8%～10% inoculum size of seed tank culture base, be cultured to logarithmic phase, the used mass percent culture medium prescription of seeding tank is: sucrose 3%, NaNO 30.2%, KCl 0.05%, MgSO 40.05%, K 2HPO 40.1%, Fe 2(SO 4) 30.001%, peptone 0.05%, pH=7.0;
The seed liquor that (3) will reach logarithmic phase is inoculated into the cultivation of production jar according to 8%～10% inoculum size, and it is identical with the seed tank culture base to produce jar used substratum;
(4) air flow of sterile air is 0.45～0.60m in the culturing process of seeding tank and production jar 3/ min, stirring velocity is 200～300rmin -1, 30～35 ℃ of temperature, the whole process incubation time is 36～48h, the nutrient solution that obtains directly is distributed into liquid dosage form with plastic barrel or packing bottle;
Said degradation bacteria ZS-S-01 is deposited at Chinese typical culture collection center (CCTCC) on April 21st, 2010, culture presevation number is CCTCC M 2010095, and the Genbank accession number of the 16S rDNA of this bacterial strain is HM016874.
3. the application of pyrethroids pesticide degradation bacterium as claimed in claim 1 aspect degradation of pyrethroid pesticide residue.
4. application as claimed in claim 3 is characterized in that described pyrethroid pesticide is fenvalerate, Deltamethrin, beta_cypermethrin or betacyfluthrin.
CN2010102841003A 2010-09-15 2010-09-15 Pyrethroid pesticide degrading bacteria and bactericide thereof Expired - Fee Related CN101974453B (en)

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CN102604859B (en) * 2012-01-17 2013-05-01 华南农业大学 Pyrethroid pesticide residue degradation bacteria as well as bacteria agent and application
CN103667146A (en) * 2013-12-12 2014-03-26 山东农业大学 DDT (2,2-bis(4-Chlorophenyl)-1,1,1-trichloroethane) and DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene) degrading bacterium DXZ9
CN106730568A (en) * 2016-11-22 2017-05-31 常州思宇知识产权运营有限公司 A kind of preparation method of biological pesticide residue degrading agent
CN107541479B (en) * 2017-09-20 2020-04-14 华南农业大学 Insecticide-esfenpropathrin degrading strain, microbial inoculum and degrading process thereof
CN110577908B (en) * 2019-09-11 2021-04-13 华南农业大学 Degradation strain of pyrethroid insecticide and application thereof

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