CN105420172B - Microbial inoculum and the application of a kind of metribuzin pesticide residual degrading bacteria and its production - Google Patents
Microbial inoculum and the application of a kind of metribuzin pesticide residual degrading bacteria and its production Download PDFInfo
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- CN105420172B CN105420172B CN201610013489.5A CN201610013489A CN105420172B CN 105420172 B CN105420172 B CN 105420172B CN 201610013489 A CN201610013489 A CN 201610013489A CN 105420172 B CN105420172 B CN 105420172B
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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
- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
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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
- A62D2101/04—Pesticides, e.g. insecticides, herbicides, fungicides or nematocides
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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 present invention provides a kind of metribuzin pesticide residual degrading bacteria and its microbial inoculums of production.The bacterial strain is gram-positive bacteria, is identified as Rhodococcus sp (Rhodococcus sp.), on June 29th, 2015 is preserved in China typical culture collection center, culture presevation number are as follows: CCTCC NO:M 2015392.It is microbial inoculum by the culture solution after the completion of QCT10 strain fermentation.QCT10 bacterial strain and its microbial inoculum can make metribuzin persticide residue in soil reduce by 50% or more, can solve metribuzin pesticide residue excessive problem in agricultural production.
Description
Technical field
The invention belongs to biological high-tech field, provide a kind of metribuzin pesticide residual degrading bacteria and its production microbial inoculum and
Using.
Background technique
The use of chemical pesticide has greatly facilitated the development of modern agriculture, has retrieved largely for agricultural production because of plant
It is lost caused by the reasons such as disease, worm, crop smothering, and greatly improves the labor efficiency and mechanization degree of agricultural production, in agriculture
Serve in the modernization of industry production immeasurable.But pesticide is some toxic and harmful substances simultaneously, they
Into after ecological environment, the biology to human hazard is not only killed, while serious harm also is caused to the ecosystem.Agriculture
A large amount of uses of medicine cause Residual Pesticides in Farm Produce exceeded, and the safety of the health and agricultural product that have seriously affected people is raw
It produces.With the improvement of people ' s living standards with the enhancing of environmental protection consciousness, the whole society directly endangers pesticide residue bring
It is also increasingly paid close attention to potential impact, there is an urgent need to the cleaning green agricultural products that non agricultural chemical residuum pollutes by people.But from present I
From the point of view of the agricultural form and demographic situation of state, to stop Pesticide use reducing farm output as the production model of cost being capable
Obstructed, continuation is also played huge work as the effective means of control phytopathy, worm, crop smothering by pesticide in agricultural production
With.This double-barreled question that how can solve in agricultural production just becomes research hotspot.Laboratory and real application research all tables
Pesticide residue in bright soil of being degraded using microorganism, so that the pesticide reduced in the agricultural product such as grain, vegetables, tealeaves is residual
It stays, the quality and economic value for improving agricultural product are feasible.
Summary of the invention
The purpose of the present invention is to provide a kind of metribuzin degradation bacteria (Rhodococcus sp.) QCT10.
Another object of the present invention is to above-mentioned metribuzin degradation bacteria QCT10 in degradation metribuzin and/or production piperazine grass
Application in ketone bacterium for degradating residual agricultural chemical.
The present invention also aims to develop a kind of novel for the practical problem and demand in production practices
Metribuzin bacterium for degradating residual agricultural chemical.
The further object of the present invention is application of the above-mentioned microbial inoculum in degradation metribuzin.
The purpose of the present invention can be achieved through the following technical solutions:
The present invention provides a kind of metribuzin degradation bacteria QCT10, is gram-positive bacteria, is identified as Rhodococcus sp
(Rhodococcus sp.), on June 29th, 2015 are preserved in China typical culture collection center, culture presevation number are as follows:
CCTCC NO:M2015392.Bacterial strain QCT10 is that pink, colony edge are smooth on LB solid plate, intermediate slightly convex.
Bacterial strain QCT10 atrichia, size are the brevibacterium (Fig. 1) of 1.6-1.7 μm of 1.1-1.2 μ m, contact enzyme positive, can be resisted mostly viscous
Rhzomorph B.Application of the metribuzin degradation bacteria QCT10 of the present invention in degradation metribuzin.
Application of the metribuzin degradation bacteria QCT10 of the present invention in production metribuzin bacterium for degradating residual agricultural chemical.
Metribuzin bacterium for degradating residual agricultural chemical is preferably prepared by the following method to obtain:
1) by above-mentioned metribuzin degradation bacteria QCT10 be inoculated in broth bouillon (10 grams of beef extract, 5 grams of peptone,
NaCl5 grams, 1000 milliliters of water, pH 7.0-7.2) in shaking flask, shaken cultivation to logarithmic phase；
2) above-mentioned cultured strain is inoculated with by the inoculum concentration of 10% (v/v) of seed tank culture base into 500 liters of seeds
Tank, the ventilatory capacity of filtrated air is 1:0.8-1.2 in incubation, and mixing speed is 180-240 revs/min, cultivation temperature 30-
32 DEG C, culture is to logarithmic growth phase, culture medium prescription used in seeding tank are as follows: glucose 1.0wt%, NH4NO31.0wt%,
K2HPO40.2wt%, MgSO40.05wt%, NaCl 0.01wt%, CaCO30.3wt%, yeast extract 0.02wt%, pH value
7.2-7.5；
3) above-mentioned seed liquor is produced into tank institute by 10% (v/v) the inoculum concentration access production tank culture of production tank culture medium
Same with culture medium and seed tank culture base phase, the ventilatory capacity of filtrated air is 1:0.8-1.2 in incubation, and mixing speed is
180-240 revs/min, cultivation temperature is 30-32 DEG C, and whole process incubation time is 36-48 hours, and thalline quantity reaches after fermentation
To 1,000,000,000/ml or more, after fermentation culture solution go out tank directly with plastic barrel or Packaging Bottle be distributed into liquid dosage form or
Solid dosage forms is distributed into packaging bag using adsorption by peat.
Beneficial effect
Had using the bacterium for degradating residual agricultural chemical that the invention produces and produces and uses at low cost, easy to use, removal effect
Good advantage is suitble to produce export base in national grain oil & vegetable or have the area of green food brand mark to be widely applied to make
With.The present invention protects the health of the people, the added value for improving agricultural product has important meaning for preserving the ecological environment
Justice.Degradation bacteria QCT10 can be by 50% or more metribuzin chemical residual degradation in soil, can be in crops using the degradation bacterial agent
Normal growth during normal use chemical pesticide carry out prevention and control of weeds and to guarantee that Residual Pesticides in Farm Produce content meets green
Color food requirement.
Present invention successfully solves metribuzin pesticide residue excessive problems in agricultural production, have both given full play to chemical agriculture
Efficient quick effect of the medicine in prevention and control of weeds, and non-toxic and non-pollution green agricultural product can be produced.
Detailed description of the invention
The transmission electron microscope of Fig. 1 degradation bacteria strains QCT10 shines
Biomaterial preservation information
A kind of metribuzin degradation bacteria QCT10, classification naming are Rhodococcus sp.QCT10, are preserved in Chinese Typical Representative training
Object collection is supported, the deposit date is on June 29th, 2015, preservation address was Wuhan, China Wuhan University, culture presevation number
Are as follows: CCTCC NO:M 2015392.
Specific embodiment
Embodiment 1: separation, the identification of bacterial strain
Acquisition by metribuzin contaminated soil, takes 15g pedotheque to be placed in the nothing of 100mL addition 50mg/L metribuzin for a long time
Machine salt fluid nutrient medium (NH4NO31.0 grams, K2HPO41.5 grams, KH2PO40.5 gram, MgSO40.2 gram, NaCl1.0 grams, water
1000 milliliters, pH 7.0-7.2) in, 30 DEG C, 150r/min culture 7 days are transferred to identical addition with 7.5% inoculum concentration
The inorganic salt liquid culture medium of 50mg/L metribuzin, continuous switching 4 times.Pass through liquid chromatography for measuring degradation effect.Take 0.5mL
The dilution pregnant solution of effective pregnant solution, gradient dilution, each gradient takes 100 μ L to be coated in containing 50mg/L metribuzin pesticide
In inorganic salts solid medium tablets, 30 DEG C of culture 5d.The single colonie grown on picking plate is inoculated in LB liquid medium (egg
1 gram of white peptone, 0.5 gram of yeast extract, NaCl0.5 grams, 20 grams of agar powder, 1000 milliliters of water, pH 7.0-7.2) in test tube, 30 DEG C,
160r/min is cultivated 1 day, is taken 1mL LB culture solution, is seeded to added with 50mgL-1In the inorganic salt liquid culture medium of metribuzin,
30 DEG C, 150r/min culture 5d, liquid chromatographic detection degradation effect.The bacterial strain for having degradation effect to metribuzin is degradation bacteria
Strain.1 plant of degradation bacteria strains is obtained by verifying, QCT10 is named as, is identified as Rhodococcus sp (Rhodococcus sp.).Bacterial strain
QCT10 is that pink, colony edge are smooth on LB solid plate, intermediate slightly convex.Bacterial strain QCT10 atrichia, size are
The brevibacterium of 1.6-1.7 μm of 1.1-1.2 μ m contacts enzyme positive, can resist polymyxin B.
Bacterial strain QCT10 is preserved in China typical culture collection center, culture presevation number on June 29th, 2015 are as follows:
CCTCC NO:M 2015392。
Degradation effect of 2 bacterial strain of embodiment to metribuzin
Degradation bacteria QCT10 measures metribuzin degradation characteristic in inorganic salt liquid culture medium: picking QCT10 single bacterium is fallen within
In 50ml LB liquid medium, 30 DEG C, 160r/min shaken cultivation 12h obtain fresh bacterium solution.By the cultured fresh bacterium of 3ml
Liquid is centrifuged 5min through 6000r/min, discards supernatant liquid, and 3ml sterile water is added and is resuspended, the bacterium solution after suspending for centrifugation.In 20ml
The metribuzin containing 50mg/l GMSM fluid nutrient medium (3.0 grams of glucose, K2HPO41.5 grams, KH2PO40.5 gram, NaCl 1.0
Gram, MgSO4·7H20.2 gram of O, 1000 milliliters of water, pH 7.0-7.2) in, the bacterium solution after access 1.5ml centrifugation suspension, 30 DEG C,
150r/min culture, is measured by sampling the content of metribuzin on the 7th day.It the results are shown in Table 1, degradation rate of the degradation bacteria QCT10 to metribuzin
Reach 75.3%.
Degraded in soil bacterium QCT10 measures metribuzin degradation characteristic: weighing 600g soil, metribuzin is added, makes soil
In concentration containing metribuzin be 15mg/kg, be added the fresh bacterium solution of QCT10, mix, make the QCT10 concentration 10 in soil11It is a thin
Born of the same parents/gram soil are placed in 30 DEG C of incubators constant temperature incubation under dark condition, the residual of metribuzin in the soil are measured by sampling within the 30th day
Amount the results are shown in Table 1, and degraded in soil bacterium QCT10 degradation metribuzin reaches 56.1%.
1 bacterial strain QCT10 of table is in GMSM fluid nutrient medium and in soil to the degradation effect of metribuzin
The preparation of 3 microbial inoculum of embodiment and degradation effect to metribuzin
The original seed of pesticide residual degrading bacteria QCT10 of the invention is activated on the test tube slant LB.By test tube slant strain
It is inoculated in broth bouillon containing 200ml (10 grams of beef extract, 5 grams of peptone, NaCl5 grams, 1000 milliliters of water, pH 7.0-7.2)
In 1000ml shaking flask, 30 DEG C of constant-temperature shaking cultures to logarithmic phase prepare inoculation seeding tank.500 liters of seeding tank, 400 liters of inventory,
Culture medium prescription (mass percent): glucose 1.0wt%, NH4NO31.0wt%, K2HPO40.2wt%, MgSO4
0.05wt%, NaCl 0.01wt%, CaCO30.3%, yeast extract 0.02%, pH value 7.2-7.5.121 DEG C of height after feeding intake
Moist heat sterilization is pressed, sterilizing is completed after being cooled to 33 DEG C, and above-mentioned cultured shaking flask strain is pressed 10% (percent by volume, similarly hereinafter)
Inoculum concentration be inoculated with into 500 liters of seeding tanks, culture to logarithmic growth phase, the control of seeding tank temperature is in 30-32 DEG C, mixing speed
220 revs/min, filtrated air intake is 1:0.8.It produces tank used medium ingredient and seed tank culture base phase is same.Produce tank
5 tons of capacity, 4.5 tons of inventory.Production tank 1.1kg/cm after feeding intake2Pressure under, 121 DEG C of high pressure moist heat sterilizations, after sterilizing
After being cooled to 33 DEG C, leads to filtrated air and keep germ-free condition spare.Seed liquor in the aforementioned seeding tank for reaching logarithmic phase is pressed
10% inoculum concentration access production tank culture, the production tank temperature degree after inoculation are controlled at 30-32 DEG C, are produced in the incubation of tank
The ventilatory capacity of filtrated air is 1:0.8, and mixing speed is 220 revs/min, and entire process flow incubation time is 48 hours.Fermentation
After thalline quantity reach 1,000,000,000/ml or more.Culture solution goes out tank and directly uses plastic barrel or Packaging Bottle after fermentation
It is distributed into liquid dosage form or solid fungicide dosage form is distributed into packaging bag using adsorption by peat.
QCT10 liquid degradation microbial inoculum measures metribuzin degradation effect in soil: taking 5000g that the agriculture of metribuzin is used for a long time
Field soil, metribuzin residual concentration are 4.7mg/kg, and QCT10 liquid degradation microbial inoculum manufactured in the present embodiment is added, and mix, make soil
QCT10 concentration in earth is 108A cell/gram soil is placed in 30 DEG C, and timing sampling measures the residual quantity of metribuzin in the soil,
It the results are shown in Table 2, QCT10 liquid degradation microbial inoculum reaches 52.3% to metribuzin degradation rate in soil in soil.
2 QCT10 metribuzin degradation bacterial agent of table is in the soil to the degradation effect of metribuzin
| Culture medium | Soil |
| Degradation rate | 52.3% |
Claims (7)
1. a kind of metribuzin degradation bacteria Rhodococcus sp. QCT10, on June 29th, 2015 are preserved in Chinese Typical Representative culture
Collection, culture presevation number are as follows: CCTCC NO:M 2015392.
2. application of the metribuzin degradation bacteria QCT10 described in claim 1 in degradation metribuzin.
3. application of the metribuzin degradation bacteria QCT10 described in claim 1 in production metribuzin bacterium for degradating residual agricultural chemical.
4. a kind of microbial inoculum prepared by metribuzin degradation bacteria QCT10 described in claim 1.
5. microbial inoculum according to claim 4, it is characterised in that be prepared by the following method:
1) metribuzin degradation bacteria QCT10 described in claim 1 is inoculated in broth bouillon shaking flask, shaken cultivation to logarithm
Phase；
2) by above-mentioned cultured strain by the 10%(v/v of seed tank culture base) inoculum concentration be inoculated with into 500 liters of seeding tanks, training
The ventilatory capacity of filtrated air is 1:0.8-1.2 during supporting, and mixing speed is 180-240 revs/min, and cultivation temperature is 30-32 DEG C,
Culture is to logarithmic growth phase, culture medium prescription used in seeding tank are as follows: glucose 1.0 wt%, NH4NO31.0 wt %, K2HPO4
0.2 wt %, MgSO40.05 wt %, NaCl 0.01 wt %, CaCO30.3 wt %, 0.02 wt % of yeast extract, pH value
7.2-7.5；
3) by above-mentioned seed liquor by the 10%(v/v of production tank culture medium) inoculum concentration access production tank culture, the training used of production tank
It supports base and above-mentioned seed tank culture base phase is same, the ventilatory capacity of filtrated air is 1:0.8-1.2 in incubation, and mixing speed is
180-240 revs/min, cultivation temperature is 30-32 DEG C, and whole process incubation time is 36-48 hours, and thalline quantity reaches after fermentation
To 1,000,000,000/ml or more, after fermentation culture solution go out tank directly with plastic barrel or Packaging Bottle be distributed into liquid dosage form or
Solid dosage forms is distributed into packaging bag using adsorption by peat.
6. application of the microbial inoculum described in claim 4 or 5 in degradation metribuzin.
7. application according to claim 6, it is characterised in that microbial inoculum metribuzin pesticide residue in degradation soil
Using.
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| CN107699523B (en) * | 2017-11-03 | 2020-06-05 | 南京农业大学 | Bacterium capable of degrading pesticides buprofezin and bifenthrin and microbial inoculum produced by bacterium |
| CN108130288B (en) * | 2017-12-15 | 2020-10-09 | 浙江工业大学 | Rhodococcus ruber and application thereof in degrading organic pollutants |
| 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 |
| CN112920971B (en) * | 2021-03-17 | 2022-11-11 | 青海省农林科学院 | Fluorochloridone degrading strain and application thereof |
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| JP4675695B2 (en) * | 2005-06-28 | 2011-04-27 | 独立行政法人農業環境技術研究所 | A novel microorganism with the ability to resolve methylthiolated triazines |
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