CN101724585B - Rhodobacter strain and application thereof - Google Patents
Rhodobacter strain and application thereof Download PDFInfo
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- CN101724585B CN101724585B CN2009100447743A CN200910044774A CN101724585B CN 101724585 B CN101724585 B CN 101724585B CN 2009100447743 A CN2009100447743 A CN 2009100447743A CN 200910044774 A CN200910044774 A CN 200910044774A CN 101724585 B CN101724585 B CN 101724585B
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Abstract
The invention provides rhodobacter strain and application thereof. The rhodobacter strain is collected in the China Center for Type Culture Collection (CCTCC), and the collection number of the rhodobacter strain is CCTCC M 20239. The rhodobacter strain can be used for preparing a pyrethroids pesticide degradation fungicide and has the advantages of low production cost, good pesticide removal effect, high efficiency, no toxicity, environmental protection, and the like. The rhodobacter strain can prepare the pyrethroids pesticide degradation fungicide through a simple, quick and low cost operation method.
Description
Technical field
The present invention relates to a kind of bacterium and application thereof, relate in particular to a kind of bacterial isolates and the application in degrading pesticide residues thereof.
Background technology
The loss that causes because of the insect pest reason has in a large number been retrieved in the agriculture prodn that is used for of chemical insecticide, has promoted the development that modern agriculture is produced greatly.But chemical insecticide has also killed beneficial organism when killing off the insect pests, and remains in the ecosystem, and the ecosystem has been caused serious harm.A large amount of uses of chemical insecticide also cause exceeding standard of pesticide residue in the agricultural-food easily, have a strong impact on the quality of agricultural-food, and people healthy brought serious threat, and the outlet of agricultural-food is produced negative influence.
Chrysanthemum ester insecticide is the compound of one type of synthetic, similar natural pyrethrin.After the eighties in 20th century, be widely used in various crop such as grain, fruit and vegetables in China.The investigation statistics of the Ministry of Agriculture shows that such agricultural chemicals is one of three main in present China outlet vegetables, fruit pesticide residues.European Union's 2007/12/EC trace standard of pesticide reaches 227, and wherein the detectability of regulation pyrethrin is 0.05mg/Kg, and standard has improved hundreds of times, causes China's tealeaves to European Union's export volume drop by half.Japan put into effect " positive list system " in 2006, and wherein the detectability of regulation pyrethrin is 2mg/Kg, made China's tealeaves export to Japan descend about 50%.The foreign exchange earning and the international competitiveness of agricultural products in China have been seriously influenced.And, the enhancing of Along with people's growth in the living standard and environmental protection consciousness, the whole society more and more pays close attention to the environmental problem that pesticide residue are brought, and people press for the green food that non agricultural chemical residuum pollutes.But; Agriculture prodn present situation and demographic situation from present China; The green agricultural product that is used for cost production non agricultural chemical residuum to stop agricultural chemicals is impracticable, and agricultural chemicals also will continue the irreplaceable effect of performance in agriculture prodn as the effective means of the sick Chinese caterpillar fungus of control.How solving this double-barreled question in the agriculture prodn becomes the research focus of each subject.
Laboratory and field practical application all show; The pesticide residual degrading bacteria technology that mikrobe is repaired to theoretical basis provides feasible approach for the pesticide residue that reduces in agricultural-food and the agriculture production environment; Characteristics efficient, nontoxic, non-secondary pollution that this technology has; And economical and practical, easy and simple to handle, become a kind of important method of removing pesticide residual contamination at present.Yet, prepare microbial inoculum easy to use, and use with rationalizing at the bacterial strain that screens, isolates the efficient degradation agricultural chemicals in a large number in the mikrobes, this all is the technical barrier that those skilled in the art need face.
Summary of the invention
The technical problem that the present invention will solve is the deficiency that overcomes prior art; Provide that a kind of production cost is low, the microorganism strains of pesticides removal effect, efficient, nontoxic, environmental protection; Correspondingly; A kind of application of microorganism strains also is provided, make its can with low-cost, conveniently operation realizes the effective degraded to pyrethroid pesticide.
For solving the problems of the technologies described above; The technical scheme that the present invention proposes is a kind of red bacterium (Rhodobacter sp.) bacterial strain, it is characterized in that: said bacterial strain is the bacterial strain of CCTCCM 209239 for being preserved in Chinese typical culture collection center (being called for short CCTCC) and preserving number.The depositary institution of this bacterial strain is Chinese typical culture collection center C CTCC, and the depositary institution address is positioned at Chinese Wuhan Wuhan University, and preservation date is on October 23rd, 2009, and this bacterial strain is named as red bacterium S2-1 (Rhodobacter sp.S2-1).
The Rhodobacter strain of the invention described above is to obtain through the screening of following method: the mud 2.0g that will pick up from certain insecticide factory adds in the 120mL PSB liquid nutrient medium, behind the illumination cultivation 7d, gets 1% and is inoculated into and contains in the Fenvalerate 100mg/L PSB liquid nutrient medium; 30 ± 2 ℃, about 3000lx is behind the illumination cultivation 7d; Get 300 μ L bacterium liquid dilutions and be applied to and contain in the Fenvalerate 100mg/LPSB solid medium, illumination cultivation to bacterium colony occurs, the bacterium that the picking colony form is different; Be inoculated into respectively and contain in the Fenvalerate 100mg/L selected liq substratum; 30 ± 2 ℃, about 3000lx behind the illumination cultivation 7d, get 1% and are inoculated into respectively in the 200mg/LPSB liquid nutrient medium; Repeat above step, in 1000mg/L PSB liquid nutrient medium.Detect pesticide concentration behind the 15d, to contain the Fenvalerate of same concentrations, the same culture conditions not substratum of inoculated bacteria is contrast.Select the maximum Rhodobacter strain (called after Rhodobacter sp.S2-1) of degradation rate to carry out follow-up study.This inoculation is cultivated in 120mL PSB substratum, 30 ± 2 ℃, about 3000lx, illumination cultivation, the gradient dilution plating method is measured the bacterial strain living weight, and bacterial concentration is adjusted to about 10 in the subsequent experimental
9Cfu/mL.
In the technique scheme, said red bacterium CCTCC M 209239 is preferably the bacterial strain with following principal character:
G-, individual morphology is shaft-like, the V-P reaction negative, clark and Lubsreaction is negative, can not utilize starch, H
2The S reaction negative can not be grown among the 3%NaCl, and optimum growth temperature is 30 ℃～35 ℃; The righttest growth pH value is 6.5～7.5; The charateristic avsorption band of thalline is 525nm, 805nm and 865nm, and 16S rDNA sequence length is 1416bp, and the accession number in Genbank is GQ429219.
Satisfy the better effects if of red bacterium CCTCC M 209239 in following application of above-mentioned feature description.
The present invention also provides a kind of above-mentioned red bacterium CCTCC M 209239 application in the degradation of pyrethroid agricultural chemicals.
Above-mentioned application is meant that preferably its concrete preparation method preferably includes following steps with said red bacterium CCTCC M 209239 preparation pyrethroids pesticide degradation bacterium agent:
A. the very low temperature conserving species of said red bacterium CCTCC M 209239 is cultivated by plating method, be cultured to red bacterium colony and occur;
B. the single bacterium colony in the said red bacterium colony is inserted serum bottle, be cultured to logarithmic phase with the Rhodobacter strain seed culture medium;
C. the bacterium liquid that obtains after step b being cultivated is produced the inoculum size (preferred 1%～5%) of substratum TV 1% and is inoculated into and produces bottle (the production bottle of 5L for example by being no less than Rhodobacter strain; This bottle can be clear-glass bottle) in, with Rhodobacter strain production culture medium culturing to logarithmic phase;
D. the bottle outlet packing makes the pyrethroids pesticide degradation bacterium agent.
In the application of above-mentioned Rhodobacter strain CCTCC M 209239, said plating method preferably adopts double-deck PSB solid medium to cultivate, and the staple of said double-deck PSB solid medium comprises (NH
4)
2SO
40.1%, MgSO
40.02%, NaHCO
30.5%, K
2HPO
40.05%, NaCl 0.02%, yeast extract paste 0.15% and agar 1.5%.
In the application of above-mentioned Rhodobacter strain CCTCC M 209239, said Rhodobacter strain seed culture medium, Rhodobacter strain are produced substratum and all are preferably the PSB liquid nutrient medium, and the staple of said PSB liquid nutrient medium comprises: (NH
4)
2SO
40.1%, MgSO
40.02%, NaHCO
30.5%, K
2HPO
40.05%, NaCl 0.02% and yeast extract paste 0.15%.
Preparing method in the above-mentioned application is through continuing to optimize the optimum culture scheme that culture condition draws; Utilize this method to cultivate Rhodobacter strain of the present invention; Have following two advantages: the one, strain growth speed is fast, and the 2nd, cheap, the 3rd, cultural method is simple, easy handling.
Preparing method's in the above-mentioned application the whole process time was generally about 25 days, after cultivate finishing, thalline quantity can reach 1,000,000,000/more than the ml, cultivate accomplish after bottle outlet can directly be distributed into liquid bacterial agent with plastic packets.
In the application of above-mentioned red bacterium CCTCC M 209239; In the culturing process each time of said step a, step b, step c; Preferred pH value is 7.0～7.2, and preferred culture temperature is 30 ℃～35 ℃, and preferred illumination condition is 2000Lx～3000Lx.Processing parameter after the aforementioned optimization can make Rhodobacter strain growth of the present invention rapider, thereby shortens the PT.
Compared with prior art; The invention has the advantages that: the invention provides that a kind of production cost is low, pesticides removal is effective, the red bacterium CCTCC M 209239 of efficient, nontoxic, environmental protection; This bacterial strain can be through simply, fast, working method prepares the pyrethroids pesticide degradation bacterium agent cheaply; It is low, easy to use that the degradation bacterial agent that uses bacterial strain of the present invention to prepare has a production cost; Advantages such as removal effect is good can be effective to the production of non agricultural chemical residuum grain, fruit, vegetables etc. in the agriculture prodn.Experiment shows; Bacterial strain of the present invention not only can be used for the degraded of substratum middle and high concentration pyrethroid pesticide (for example Fenvalerate) under the laboratory condition, also can be used for the degraded and the removal (PP-383 of for example removing on the Chinese cabbage is residual) of pyrethroid remains of pesticide on the farm crop of field.Application of the present invention can normally used chemical pesticide, eliminating pest and then guarantee under the prerequisite of crop yield to farm crop, can make pesticide residue in the farm crop such as grain, fruit, vegetables reduce to very low or do not have again.And bacterial strain of the present invention itself has multiple nutritional components and active substance, can effectively promote plant growth, has the effect that prevents and treats crop pest concurrently, has characteristics safe, nontoxic, environmental protection.In addition, because bacterial strain of the present invention can be applied to the degrading high concentration pyrethroid pesticide, therefore also can be used for the processing of pesticide wastewater.
A kind of Rhodobacter strain (Rhodobacter sp.S2-1), its depositary institution is Chinese typical culture collection center (being called for short CCTCC), and preserving number is CCTCC M 209239, and preservation date is on October 23rd, 2009.
Embodiment
Embodiment 1:
A kind of Rhodobacter strain of the present invention, this bacterial strain are preserved in Chinese typical culture collection center C CTCC, and preserving number is CCTCC M 209239.This bacterial strain has following principal character:
G-, individual morphology is shaft-like, the V-P reaction negative, clark and Lubsreaction is negative, can not utilize starch, H
2The S reaction negative can not be grown among the 3%NaCl, and optimum growth temperature is 30 ℃～35 ℃; The righttest growth pH value is 6.5～7.5; The charateristic avsorption band of thalline is 525nm, 805nm and 865nm, and 16S rDNA sequence length is 1416bp, and the accession number in Genbank is GQ429219.
Present embodiment is with above-mentioned red bacterium CCTCC M 209239 preparation pyrethroids pesticide degradation bacterium agent, and its method specifically may further comprise the steps:
(1) activation: the very low temperature conserving species of above-mentioned red bacterium CCTCC M 209239 is cultivated by the double-layer plate culture method, and this double-layer plate culture method specifically is to cultivate with double-deck PSB solid medium, and its staple comprises (NH
4)
2SO
40.1%, MgSO
40.02%, NaHCO
30.5%, K
2HPO
40.05%, NaCl 0.02%, yeast extract paste 0.15% and agar 1.5%, the pH value during cultivation is 7.0～7.2, and temperature is 30 ℃～35 ℃, and illumination condition is 2000Lx～3000Lx, is cultured to red bacterium colony and occurs;
(2) seed culture: the single bacterium colony in the red bacterium colony that step (1) is turned out inserts serum bottle, and with the cultivation of PSB liquid nutrient medium, the staple of this PSB liquid nutrient medium comprises: (NH
4)
2SO
40.1%, MgSO
40.02%, NaHCO
30.5%, K
2HPO
40.05%, NaCl 0.02% and yeast extract paste 0.15%, the pH value during cultivation is 7.0～7.2, and temperature is 30 ℃～35 ℃, and illumination condition is 2000Lx～3000Lx, is cultured to logarithmic phase;
(3) produce to cultivate: 1%～5% the inoculum size that the bacterium liquid that obtains after step (2) is cultivated is produced the substratum TV by Rhodobacter strain is inoculated into produces in the bottle, and with Rhodobacter strain production culture medium culturing, the staple of this production substratum comprises: (NH
4)
2SO
40.1%, MgSO
40.02%, NaHCO
30.5%, K
2HPO
40.05%, NaCl 0.02% and yeast extract paste 0.15%, the pH value during cultivation is 7.0～7.2, and temperature is 30 ℃～35 ℃, and illumination condition is 2000Lx～3000Lx, is cultured to logarithmic phase;
(4) the bottle outlet packing makes the pyrethroids pesticide degradation bacterium agent.
The whole process incubation time of present embodiment method is 25 days, after cultivate finishing, thalline quantity reach 1,000,000,000/more than the ml.
Embodiment 2:
Bacterial strain of the present invention is in the application of degraded substratum middle and high concentration Fenvalerate
Fenvalerate is used acetone solution, add in the experiment substratum to the final concentration of Fenvalerate be 600mg/L, used experiment substratum is: (NH
4)
2SO
40.1%, MgSO
40.02%, NaHCO
30.5%, K
2HPO
40.05%, NaCl 0.02% and yeast extract paste 0.15%, the pH value of this experiment substratum is 7.0～7.2, and temperature is 30 ℃～35 ℃, and intensity of illumination is 2000Lx～3000Lx, the microbial inoculum for preparing among the embodiment 1 is inoculated in this experiment substratum cultivated 15 days.Cultivate the Fenvalerate in the back experiment substratum with petroleum ether extraction, it is residual that gas chromatograph (Agilent 6890N) is measured Fenvalerate, and detector is electron capturer (ECD).Being not contrast with the experiment substratum of microbial inoculum.All handle triplicate, and measurement result sees the following form shown in 1.
Table 1: Fenvalerate degradation rate contrast in the experiment substratum of present embodiment 2
Visible by last table 1, use bacterial strain of the present invention, can make the degradation rate of 600mg/L Fenvalerate reach 37.34% in 15 days.
Embodiment 3:
The application of bacterial strain of the present invention PP-383 in degraded field Chinese cabbage and soil
Select for use 4.5% r (production of Nanjing red sun Group Plc) as medicament, recommend the using dosage upper limit 1.5 times, spray the 60kg water yield by every mu and calculate spraying, chemicals treatment sub-district area is 32.25m
2, control treatment sub-district area is 80.75m
2, the interval (10 days) of gather interval (14 days) and twice dispenser of recommending during according to registration, confirm dispenser three times.Spraying time is August 31 for the first time, is September 10 for the second time, is September 20 for the third time.
Get the microbial inoculum 200ml of embodiment 1 preparation, evenly spray on Chinese cabbage (Xiayang is white) after converting water 60kg.For the first time spraying the microbial inoculum time is August 31, is September 10 for the second time, is September 20 (all after medicament sprays, spraying microbial inoculum) for the third time.With the field Chinese cabbage that sprays microbial inoculum as contrast.All handle triplicate.
Sampling on October 6.
The plant sample: 4 samplings of sub-district diagonal method, every point sampling 1 strain in every processing sub-district, ill, excessive, too small plant is not all adopted, and mashed leaf and Huang Ye are removed in totally 4 strains of every processing sub-district.Earlier with the vertical quadrisect of every strain Chinese cabbage, to get relative two parts and be cut into the fragment that is about 2cm again, 4 strains are fully mixed thoroughly after all cutting, and then get two parts of relative synthetic plant samples that about 1kg weighs with quartering again if surpass 1kg.The whole process operation personnel that take a sample, cut appearance and dress appearance all wear the disposable plastic gloves, and other utensils of dress sample all fill up plastics film, change gloves and plastics film after each processing sampling is accomplished.
Pedotheque: 4 samplings of sub-district diagonal method, every processing sub-district are gathered about 0～10cm topsoil soil sample 1Kg; Control sample is handled on the no dish clear area of sub-district at each, and 4 samplings of diagonal method are gathered about 0～10cm topsoil soil sample 1kg and are mixed into a sample.The sampling operation personnel wear the disposable plastic gloves, and each is handled and changes disposable plastic gloves and trier after sampling is accomplished.
All samples all send Hunan Province's agricultural product quality to detect the inspection center November 4 the same year and detects.Detected result sees the following form 2.
Table 2: the field Chinese cabbage of present embodiment 3 and the contrast of the PP-383 degradation rate in the soil
Can know that by last table 2 in the practical application of field, can make that the degradation rate of PP-383 reaches 62.07% in the Chinese cabbage, the degradation rate of PP-383 reaches 51.86% in the soil.
Claims (7)
1. Rhodobacter strain is characterized in that: said red bacterium (Rhodobacter sp.) bacterial strain is to be preserved in Chinese typical culture collection center and preserving number is the bacterial strain of CCTCC M 209239.
2. the application of Rhodobacter strain in the degradation of pyrethroid agricultural chemicals according to claim 1.
3. application according to claim 2 is characterized in that, prepares the pyrethroids pesticide degradation bacterium agent with said Rhodobacter strain, and its concrete preparation method may further comprise the steps:
A. the conserving species of said Rhodobacter strain is cultivated by plating method, be cultured to red bacterium colony and occur;
B. the single bacterium colony in the said red bacterium colony is inserted serum bottle, be cultured to logarithmic phase with the Rhodobacter strain seed culture medium;
C. the bacterium liquid that obtains after step b being cultivated is produced the inoculum size of substratum TV 1% and is inoculated into and produces in the bottle by being no less than Rhodobacter strain, with Rhodobacter strain production culture medium culturing to logarithmic phase;
D. the bottle outlet packing makes the pyrethroids pesticide degradation bacterium agent.
4. application according to claim 3 is characterized in that: said plating method is to adopt double-deck PSB solid medium to cultivate, and the staple of said double-deck PSB solid medium comprises (NH
4)
2SO
40.1%, MgSO
40.02%, NaHCO
30.5%, K
2HPO
40.05%, NaCl 0.02%, yeast extract paste 0.15% and agar 1.5%.
5. application according to claim 3 is characterized in that: said Rhodobacter strain seed culture medium, Rhodobacter strain are produced substratum and are the PSB liquid nutrient medium, and the staple of said PSB liquid nutrient medium comprises: (NH
4)
2SO
40.1%, MgSO
40.02%, NaHCO
30.5%, K
2HPO
40.05%, NaCl 0.02% and yeast extract paste 0.15%.
6. according to claim 3,4 or 5 described application; It is characterized in that: in the culturing process each time of said step a, step b, step c; The pH value all is controlled to be 7.0～7.2, and culture temperature all is controlled to be 30 ℃～35 ℃, and illumination condition all is controlled to be 2000Lx～3000Lx.
7. according to claim 3,4 or 5 described application, it is characterized in that: the inoculum size of bacterium liquid is 1%～5% of a Rhodobacter strain production substratum among the said step c.
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| CN2009100447743A CN101724585B (en) | 2009-11-19 | 2009-11-19 | Rhodobacter strain and application thereof |
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| CN101724585B true CN101724585B (en) | 2012-01-25 |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107674843B (en) * | 2017-08-15 | 2020-09-25 | 云南万魁生物科技有限公司 | Rhodobacter xylinum strain RC1 and application thereof |
| CN110283738B (en) * | 2019-05-22 | 2020-10-16 | 湖南省植物保护研究所 | Photosynthetic bacterium capsular rhodobacter strain, microbial inoculum medicament, and preparation method and application thereof |
| CN110205262B (en) * | 2019-05-22 | 2020-10-16 | 湖南省植物保护研究所 | Rhodobacter sphaeroides A2 strain, biocontrol microbial inoculum and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101139559A (en) * | 2006-09-05 | 2008-03-12 | 许雷 | Novel bacterial strain for highly effective degradation of chrysanthemum ester and organophosphorus pesticide and uses thereof |
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2009
- 2009-11-19 CN CN2009100447743A patent/CN101724585B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101139559A (en) * | 2006-09-05 | 2008-03-12 | 许雷 | Novel bacterial strain for highly effective degradation of chrysanthemum ester and organophosphorus pesticide and uses thereof |
Non-Patent Citations (3)
| Title |
|---|
| 张战泓 等.一株光合细菌分离鉴定及其降解甲氰菊酯特性.《湖南农业大学学报(自然科学版)》.2009,第35卷(第4期),全文. * |
| 张松柏 等.甲氰菊酯降解菌鉴定及其降解特性.《环境污染与防治》.2008,第30卷(第7期),全文. * |
| 张松柏 等.降解甲氰菊酯光合细菌的分离鉴定及其降解特性研究.《农业环境科学学报》.2009,第28卷(第1期),全文. * |
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