CN117603887A - Low-temperature degradation herbicide synthetic flora and application thereof - Google Patents
Low-temperature degradation herbicide synthetic flora and application thereof Download PDFInfo
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- 239000004009 herbicide Substances 0.000 title claims abstract description 142
- 230000002363 herbicidal effect Effects 0.000 title claims description 90
- 238000006731 degradation reaction Methods 0.000 title abstract description 81
- 230000015556 catabolic process Effects 0.000 title abstract description 80
- BGZZWXTVIYUUEY-UHFFFAOYSA-N fomesafen Chemical compound C1=C([N+]([O-])=O)C(C(=O)NS(=O)(=O)C)=CC(OC=2C(=CC(=CC=2)C(F)(F)F)Cl)=C1 BGZZWXTVIYUUEY-UHFFFAOYSA-N 0.000 claims abstract description 24
- WVQBLGZPHOPPFO-UHFFFAOYSA-N 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(1-methoxypropan-2-yl)acetamide Chemical compound CCC1=CC=CC(C)=C1N(C(C)COC)C(=O)CCl WVQBLGZPHOPPFO-UHFFFAOYSA-N 0.000 claims abstract description 23
- VTNQPKFIQCLBDU-UHFFFAOYSA-N Acetochlor Chemical compound CCOCN(C(=O)CCl)C1=C(C)C=CC=C1CC VTNQPKFIQCLBDU-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000005586 Nicosulfuron Substances 0.000 claims abstract description 23
- HKPHPIREJKHECO-UHFFFAOYSA-N butachlor Chemical compound CCCCOCN(C(=O)CCl)C1=C(CC)C=CC=C1CC HKPHPIREJKHECO-UHFFFAOYSA-N 0.000 claims abstract description 23
- RTCOGUMHFFWOJV-UHFFFAOYSA-N nicosulfuron Chemical compound COC1=CC(OC)=NC(NC(=O)NS(=O)(=O)C=2C(=CC=CN=2)C(=O)N(C)C)=N1 RTCOGUMHFFWOJV-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000005583 Metribuzin Substances 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 22
- FOXFZRUHNHCZPX-UHFFFAOYSA-N metribuzin Chemical compound CSC1=NN=C(C(C)(C)C)C(=O)N1N FOXFZRUHNHCZPX-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000005472 Bensulfuron methyl Substances 0.000 claims abstract description 20
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- 230000000593 degrading effect Effects 0.000 claims abstract description 18
- XCSGPAVHZFQHGE-UHFFFAOYSA-N alachlor Chemical compound CCC1=CC=CC(CC)=C1N(COC)C(=O)CCl XCSGPAVHZFQHGE-UHFFFAOYSA-N 0.000 claims abstract description 8
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- PPWBRCCBKOWDNB-UHFFFAOYSA-N bensulfuron Chemical compound COC1=CC(OC)=NC(NC(=O)NS(=O)(=O)CC=2C(=CC=CC=2)C(O)=O)=N1 PPWBRCCBKOWDNB-UHFFFAOYSA-N 0.000 description 3
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 238000009328 dry farming Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C2101/00—In situ
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- C12R2001/07—Bacillus
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
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Abstract
The invention discloses a synthetic flora of low-temperature degradation herbicides and application thereof, and relates to the field of microorganisms, four functional strains capable of degrading eight herbicides, namely alachlor, acetochlor, butachlor, metolachlor, metribuzin, nicosulfuron, bensulfuron-methyl and fomesafen, at low temperature are screened and separated by a plate condition screening method, and the functional strains are named as H1, B1, K1 and Y1 respectively.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a low-temperature grass reducing agent synthetic flora and application thereof.
Background
At present, aiming at the pollution of the soil composite herbicide, the method commonly used is to degrade the residual herbicide in the soil by using physical, chemical, biological and other repair technologies. Physical repair mainly comprises a landfill method, a soil replacement method and a ventilation decontamination method, but the methods only solve the short-term problems, cannot effectively and radically treat pollution for a long time, and consume a great deal of time, energy and money. The chemical restoration is to add chemical modifier into soil, and the chemical reaction between herbicide and modifier is used to restore soil. Bioremediation is the best way to utilize the metabolic activity of microorganisms to degrade residual pesticides in soil, and is the pollution remediation and sustainable development in the current environmental field. The biological repairing method comprises the steps of repairing the biological carbon, repairing the animals and the plants, repairing the microbial inoculum and the like, wherein in the repairing method, the using amount of the biological carbon is large, the period of repairing the animals and the plants is long, and the microbial repairing operation is simple, the cost is low, and other pollution is avoided.
The northeast area is cold in winter and long, the microbial decomposition speed of the black soil is low, and the soil contains more organic matters, so that herbicide residues in the black soil are difficult to degrade due to the factors. According to different action mechanisms, the common herbicides in the northeast black soil at present comprise atrazine, acetochlor, butachlor, metribuzin, metolachlor, nicosulfuron, bensulfuron-methyl, fomesafen and the like. Typical herbicide pollution can be completely degraded through the co-degradation or synergistic effect of microorganisms, and the degradation and conversion of the herbicide has obvious correlation with the microbial flora structure and organic matter content in the soil. At present, microbial degradation of herbicide mainly focuses on single strain, and the problems of single degradation object, unstable degradation capability and the like generally exist, and the microorganism or microbial inoculum discovered at present can degrade herbicide but cannot play a good role in low temperature in northeast. The herbicide degrading strain discovered by the current research mostly degrades at medium temperature (30-35 ℃), and the strain which can degrade herbicide at low temperature is less.
Therefore, the strain capable of efficiently degrading and removing the herbicide in the soil is excavated, the synthetic flora is constructed, the stable degradation capability of the microbial inoculum on the herbicide is improved, and the method has important significance for the farmland soil environmental health and sustainable development of northeast black lands.
Disclosure of Invention
Aiming at the situation, in order to overcome the defects of the prior art, the invention provides a low-temperature degradation herbicide synthetic flora and application thereof, and the related low-temperature degradation herbicide synthetic flora comprises pseudomonas with the preservation number of CGMCC No.29205Pseudomonas sp.) B1, acinetobacter with preservation number of CGMCC No.29206Acinetobacter sp.) K1 bacillus with preservation number of CGMCC No.29207Bacillus sp.) H1 and comamonas with preservation number of CGMCC No.29208Comamonas sp.) Y1, the constructed synthetic flora is 'Y1+K1+B1' and 'H1+K1+B1' (hereinafter simply referred to as YBK/HBK respectively), can realize high-efficiency degradation of eight herbicides under low temperature condition, and improves the ecological function of residual composite herbicide soil.
The inventor utilizes a plate condition screening method to screen and separate four functional strains capable of degrading eight herbicides of alachlor, acetochlor, butachlor, metolachlor, metribuzin, nicosulfuron, bensulfuron and fomesafen at low temperature, and the eight functional strains are respectively identified as pseudomonas after biochemical identificationPseudomonas sp.) B1, acinetobacter @Acinetobacter sp.）K1、Bacillus [ ]Bacillus sp.) H1 and Comamonas spComamonas sp.) Four strains Y1, and preserving the four strains respectively; hereinafter, the four strains are respectively abbreviated as: b1, K1, H1, Y1; the basic properties of the strains are shown in Table 1.
Specifically, the invention is realized through the following scheme:
in a first aspect, the invention provides two synthetic flora, namely HBK synthetic flora formed by three strains of H1, B1 and K1 and YBK synthetic flora formed by three strains of Y1, B1 and K1, wherein the two synthetic flora can completely degrade 250mg/L composite herbicide (more than 99.2%), and the degradation capability is obviously better than that of single strains B1, K1, H1 and Y1, and the degradation effect of the combination of strains (Y1 +B1, Y1+K1, K1+B1, H1+K1 and K1 +B1) is obviously better than that of the combination of strains (Y1 +B1, Y1+K1, H1+K1 and K1).
Preferably, in the HBK synthetic flora, the ratio of the total viable count of the three strains H1, B1, K1 is 1:1:1, a step of; in YBK synthetic flora, the total viable count ratio of the three strains Y1, B1 and K1 is 1:1:1.
in a second aspect, the invention provides a composite microbial inoculant comprising the synthetic flora of the previous aspect.
Preferably, the composite microbial inoculum is prepared by mixing seed solutions of single bacteria in equal proportion.
In a third aspect, the present invention provides the use of a synthetic flora as in the first aspect or a complex inoculant as in the second aspect, for degrading at least one of atrazine, acetochlor, butachlor, metolachlor, metribuzin, nicosulfuron, bensulfuron-methyl and fomesafen herbicide.
In a fourth aspect, the present invention provides the use of a synthetic flora as in the first aspect or a complex inoculant as in the second aspect, for degrading at least one of atrazine, acetochlor, butachlor, metolachlor, metribuzin, nicosulfuron, bensulfuron-methyl and fomesafen herbicide remaining in the soil.
In a fifth aspect, the present invention provides the use of the synthetic flora in the first aspect or the complex inoculant in the second aspect, for degrading at least one of atrazine, acetochlor, butachlor, metolachlor, metribuzin, nicosulfuron, bensulfuron-methyl and fomesafen herbicides in a low temperature environment.
Alternatively, in the above-mentioned use, the low-temperature environment has a temperature in the range of-5 to 20 ℃.
In a sixth aspect, the present invention provides the use of the synthetic flora of the first aspect or the complex inoculant of the second aspect, to degrade at least one of the eight herbicides remaining in the soil, atrazine, acetochlor, butachlor, metolachlor, metribuzin, nicosulfuron, bensulfuron-methyl and fomesafen at low temperature.
Alternatively, among the above uses, the use is to degrade herbicides remaining in soil, including triazines (atrazine, zinone), amides (acetochlor, butachlor, metolachlor), sulfonylureas (nicosulfuron, bensulfuron) and grass ethers (fomesafen), and the degradation ability of eight herbicides at different residual concentrations in soil in different areas is very considerable.
Alternatively, in the application, the temperature condition of the low-temperature environment is 10 ℃, at the temperature, the composite herbicide in the soil is removed, the degradation temperature range of the composite herbicide is lower than the degradation temperature range (mostly the degradation at the medium temperature of 30-35 ℃) of herbicide degradation strains which are found by current researches, namely the composite herbicide can degrade the herbicide in the soil under the low-temperature condition, and the composite herbicide has good application prospect especially in removing herbicide pollution in black soil in northeast areas.
In a seventh aspect, the present invention provides the synthetic flora of the first aspect and the complex inoculant of the second aspect, for use in improving the ecological function of residual herbicide soil.
Alternatively, in the above application, the residual herbicide in the soil may comprise eight herbicides mentioned in the third aspect, a higher herbicide degradation rate is ensured for the soil with residual composite herbicide, and after detection, the synthetic flora of the first aspect or the composite microbial inoculum of the second aspect is added into the soil, so that the ecological function of the soil is significantly improved.
In an eighth aspect, the present invention provides a method for improving the ecological function of residual composite herbicide soil, the method comprising applying an effective amount of the synthetic flora of the first aspect or the composite microbial inoculum of the second aspect to the soil, stirring the soil and the soil thoroughly, mixing the soil uniformly, increasing the content of beta-galactosidase, alpha-glucosidase, beta-glucosidase, urease, protease, phosphatase and dehydrogenase in the soil, decreasing malondialdehyde and catalase, and significantly improving the ecological function of the soil.
Compared with the prior art, the invention has the following beneficial effects:
the two synthetic flora or the composite microbial inoculum can efficiently degrade eight herbicides of atrazine, acetochlor, butachlor, metolachlor, metribuzin, nicosulfuron, bensulfuron-methyl and fomesafen remained in soil under the low temperature condition, and can completely degrade 250mg/L of the eight herbicides (more than 99.2 percent) at the temperature of 4 ℃, thereby having wide application potential and value.
The two synthetic flora or the composite microbial inoculum has considerable degradation capability on the eight herbicides remained in the soil of five typical black lands under the low temperature condition, the degradation rate of the herbicides reaches more than 63%, most of the degradation rate can reach more than 85%, the contents of beta-galactosidase, alpha-glucosidase, beta-glucosidase, urease, protease, phosphatase and dehydrogenase in the soil are increased, malondialdehyde and catalase are reduced, and the ecological functions of the soil are obviously improved.
Therefore, the synthetic flora YKB and HKB related by the invention have good degradation effects on eight kinds of composite herbicides currently existing in the soil of northeast black soil, can maintain higher activity at low temperature (the activity of the synthetic flora or the composite microbial agent in degrading eight kinds of herbicides) and have no adverse effect on the environment, have higher universality, solve the problem that the existing microbial agent cannot degrade the composite herbicide at low temperature, have great application prospect in relieving and removing pollution of the residual herbicide in the soil of northeast black soil, and have important significance on the quality of the soil of the black soil farmland, the grain safety and the human health.
Biological material preservation information:
pseudomonas spPseudomonas sp.) B1 is preserved in China general microbiological culture collection center (CGMCC) with a preservation number of CGMCC No.29205, a preservation date of 2023, 12 months and 1 day, a preservation address of North Chen Xili No. 1, 3 in the Korean area of Beijing city, and the state of survival;
acinetobacter @Acinetobacter sp.) K1 is preserved in China general microbiological culture Collection center (CGMCC) No.29206, the preservation date is 2023, 12 months and 1 day, the preservation address is North Chen Xili No. 1, 3 in the Korean area of Beijing city, and the state is survival;
bacillus [ ]Bacillus sp.) H1 is preserved in China general microbiological culture Collection center (CGMCC) No.29207, the preservation date is 2023, 12 months and 1 day, the preservation address is North Chen Xili No. 1, 3 in the Korean area of Beijing city, and the state is survival;
comamonas spComamonas sp.) Y1 is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.29208, a preservation date of 2023, 12 months and 1 day, and a preservation address of North Chen Xiu No. 1, 3 in the Korean region of Beijing city, in a state of survival.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention.
FIG. 1 is a technical scheme of strain selection in example 1 of the present invention;
FIG. 2 is a colony morphology (top) and SEM electron microscopy (bottom) image of the strain selected in example 1 of the present invention (strains B1, H1, K1, Y1 in order from left to right);
FIG. 3 is a comparison of the degradation capacity of eight herbicides in the single/complex flora laboratory conditions in example 2 of the present invention;
FIG. 4 is a partial comparative graph showing the degradation ability of HBK in a soil simulation experiment for eight herbicides in five areas in example 3 of the present invention;
FIG. 5 is another partial comparative graph of HBK in the soil modeling experiment comparing the degradation ability of eight herbicides in five areas in example 3 of the present invention;
FIG. 6 is a partial comparative graph showing the degradation ability of YBK in a soil simulation experiment for eight herbicides in five regions in example 3 of the present invention;
fig. 7 is another partial comparative graph of YBK in the degradation ability comparison of eight herbicides in five areas in the soil simulation experiment in example 3 of the present invention.
Detailed Description
The invention discloses a low-temperature degradation herbicide synthetic flora and application thereof, and a person skilled in the art can properly improve process parameters by referring to the content of the low-temperature degradation herbicide synthetic flora. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
In an embodiment of the invention, the medium involved is specifically as follows: the formula of the R2A medium is as follows (1L): yeast powder 0.5g, soluble starch 0.5g, peptone 0.5g, dipotassium hydrogen phosphate 0.3g, casein amino acid 0.5g, sodium pyruvate 0.3g, glucose 0.5g, mgSO 4 ∙7H 2 O0.05 g; sterilizing at 121deg.C for 20min. The formula of the 1/10LB medium is as follows (1L): 1g of tryptone, 0.5g of yeast extract and 1g of sodium chloride, adjusting the pH of the culture medium to 7.4 by NaOH, weighing and dissolving each component of the culture medium in proportion, and sterilizing by high-pressure steam at 121 ℃ for 20min.
1/10LB solid medium is cultured in the same liquid medium, except that 1.5% of agar is added.
Example 1: (isolation of potential functional strains from soil in northeast black soil areas by means of plate condition screening) microorganisms or microbial agents currently found, although degrading herbicides, cannot exert a good effect in northeast low-temperature environments in China. The herbicide degrading strain discovered by the current research mostly degrades at medium temperature (30-35 ℃), and the strain which can degrade herbicide at low temperature is less. In addition, the inventors analyzed the characteristics of the soil in the northeast black soil region, especially the soil in five typical dry-farming regions (Liaoning, jilin, harbin, seilding, heihe), and the analysis showed that eight typical herbicides remained in the soil, including alachlor, acetochlor, butachlor, metolachlor, metribuzin, nicosulfuron, bensulfuron, and fomesafen. The eight herbicide residues lead to complex soil environment in northeast black soil areas, and particularly the environment temperature in one year in northeast areas is mostly in a low-temperature environment, which is very unfavorable for degrading the residual herbicide in the soil by utilizing the microbial agent.
Based on the current situation, the inventor screens and separates four strains from the soil in the northeast black soil area by using a plate condition screening method, and finally obtains pseudomonas after corresponding identificationPseudomonas sp.) B1, acinetobacter @Acinetobacter sp.) K1, bacillusBacillus sp.) H1 and Comamonas spComamonas sp.) Four strains Y1.
The four strains are used as antagonism experiments, the obtained four strains are prepared into synthetic flora by different combination modes, and the degradation capability of corresponding composite herbicide (high performance liquid chromatography) is measured.
The following are specific steps of the plate screening method in this embodiment:
(1) Sample collection: the soil in five typical areas of northeast areas is collected as a culture pattern, conditional screening is carried out aiming at the characteristics of the soil in the northeast areas, and the screened strain has more pertinence.
Specifically, in this example, soil from halbine, seismosis, black river, and the like, was collected in the city of iron-green, jilin, and Jielin, and from the city of Liaoning, jielin, 7 months in 2023.
(2) Sample enrichment: accurately weighing 100g of soil sample in a 250mL sterilization triangular flask, adding 200 mug/kg of alachlor, acetochlor, butachlor, metolachlor, metribuzin, nicosulfuron, bensulfuron-methyl and fomesafen, slightly stirring, placing in a 28 ℃ incubator for culturing for one week, taking care of adding sterile water, and keeping the moisture value unchanged.
(3) Isolation of strains: accurately weighing 10g of herbicide enriched soil sample, placing in a triangular flask filled with 90mL of sterile physiological saline, shaking in a shaking table at 28deg.C for one hour, taking out, standing for two hours, sequentially taking 1mL to 9mL of sterile physiological saline, gradually diluting to 10 -3 、10 -4 、10 -5 Respectively taking 0.1mL of R2A solid culture medium plates coated with eight composite herbicides (the concentration is 200 mug/kg respectively), coating 3 plates at each dilution, culturing in a 28 ℃ incubator for 5 days, growing strains which are resistant bacteria capable of resisting eight herbicides, and storing the plates in a 4 ℃ refrigerator for later use.
(4) Scribing and separating: the herbicide resistant bacteria obtained in step (3) were picked up colonies of different forms and streaked repeatedly several times to obtain pure isolates. Streaking with 1/10LB culture medium plate, and storing in glycerol tube to-80deg.C refrigerator after bacteria grow out.
(5) Screening strains with eight composite herbicide removal capacities: and (3) performing herbicide degradation capability test on each strain separated in the step (4), namely respectively inoculating each strain in eight composite herbicide 1/10LB culture mediums with the concentration of 200 mug, placing the composite herbicide 1/10LB culture mediums at the temperature of 4 ℃ for shake cultivation for 72 hours at the speed of 150rpm, sucking 1mL of culture solution, centrifuging the culture solution at the speed of 12000rpm for 1min, sucking the supernatant, filtering, taking filtrate, detecting the residual herbicide content of the solution by using a high performance liquid chromatograph, and selecting strains capable of enabling the herbicide to be removed by more than 80 percent as primary screening strains.
On the basis, four obtained bacteria are used as antagonism experiments, synthetic flora is prepared by different combination modes, the degradation capacity of corresponding composite herbicide (high performance liquid chromatography) is measured, the composite herbicide is transferred into a 1/10LB liquid culture medium, eight composite herbicides (with the concentration of 200 mug/kg respectively) are added, the composite herbicide is placed in a shaking table at the temperature of 28 ℃ for shaking culture, 2mL of bacterial liquid is taken for 12000rpm for centrifugation for 2min, and the removal of the herbicide in the supernatant is detected by high performance liquid chromatography detection.
(6) Classification and identification of strains with herbicide degradation capability: firstly, the 16SrDNA identification is carried out by adopting a prokaryotic 16SrDNA universal primer 27F (5 '-AGAGTTTGATCMTGGCTCAG-3') and a 1492R (5 '-GGYTACCTTGTTACGACTT-3'), namely, a specific PCR method is referred to as a patent of a soil total DNA small-quantity rapid extraction method (patent number: 2005101205847, public day: 7 month 4 of 2007) and sequencing, then the DNA is compared with an international NCBIGenBank (www.ncbi.nlm.nih.gov) nucleotide database, the nucleotide homology is 99%, and secondly, the morphological identification (refer to figure 2) and the growth characteristic identification are carried out by utilizing a transmission electron microscope.
Morphological features are as follows: bacterial strains Y1, K1, B1 and H1 are all yellow in colony color, can grow under the low-temperature condition (0-4 ℃), the optimal growth temperature is 28-35 ℃, and the optimal growth NaCl concentration is 0-0.5%. Physiological and biochemical analysis refer to table 1.
TABLE 1 basic characteristics table of strains
Pseudomonas spPseudomonas sp.) B1, bacillusBacillus sp.) H1, acinetobacter @Acinetobacter sp.) K1, comamonas sp @, comamonas spComamonas sp.) Y1 is cultured in 1/10LB liquid or solid medium at 28℃and can be preserved for a short period of time at 4℃after the culture.
If stored for a long period of time, the strain may be suitably stored using a glycerol freezer or freeze-dryer (see for details: zhao, he Shaojiang, microbiology experiments, first edition, science publishers. Beijing, 2002: P202-205).
Example 2: (exploration of degradation of Complex herbicide by novel synthetic flora YKB and HKB in a culture medium simulating Complex herbicide pollution at low temperature) the inventors prepared four strains obtained after screening and identification in example 1 into synthetic flora by different combination methods, and measured the degradation capacities of the corresponding complex herbicides (high performance liquid chromatography), specifically, the inventors firstly carried out degradation capacities of the single strains (H1, Y1, B1, K1) and different combination methods (Y1+B1, H1+K1, Y1+K1, H1+B1, K1+B1, Y1+K1+B1, H1+K1+B1+B1) respectively at 4 ℃, and the experimental results are referred to in Table 2 and FIG. 3.
The test results show that the removal condition of a single strain on the high-concentration herbicide is 40-70%, the degradation rate of the strain in the two combinations (Y1+B1, Y1+K1, K1+B1, H1+B1, H1+K1 and K1) is increased to 50-96%, the degradation rate of the synthetic flora YKB and HKB is the highest, 250mg/L of the composite herbicide can be completely degraded (more than 99.2%), the concentration and the removal efficiency are far higher than those of the strain/synthetic flora found in the prior study, the analysis shows that the three strains have synergistic effect, three strains in the two synthetic flora of Y1+K1+B1 and H1+K1 interact to jointly degrade eight herbicides with high concentration in the same reaction system, the degradation rate is far higher than that of the single strain, and the synthetic flora can still maintain the activity of degrading the high-concentration herbicide under the low-temperature condition.
Other factors are unchanged, for the synthetic flora YKB and HKB, the degradation rate of the high-concentration composite herbicide is detected at the reaction system temperature of minus 5 ℃, 0 ℃, 10 ℃, 15 ℃ and 20 ℃, and the detection result is shown in a table 3, and according to the table 3, the degradation rate of the two composite flora can reach 100% at the temperature of 10 ℃ and 20 ℃, and it is also expected that the activity is kept better along with the temperature rise (to the optimal growth temperature), the activity is kept to be consistent with the characteristics of the strain, the activity is reduced to a certain extent even dormant and dies after the temperature is gradually reduced, but the composite strain related to the application can still keep extremely high degradation rate at the temperature of 4-10 ℃ and is consistent with the expectation that the inventor keeps high herbicide degradation rate of the composite strain in a low-temperature environment.
In combination with the experimental results, the synthetic flora (composite microbial inoculant) can still keep high degradation rate at the temperature of 4-20 ℃, 250mg/L composite herbicide can be completely removed (more than 99.2%), and particularly, the synthetic flora (composite microbial inoculant) HKB and YKB can still keep high degradation rate (more than 50%) in a lower temperature range (-5-4 ℃), wherein the synthetic flora/composite microbial inoculant of HKB keeps 70% degradation rate in a lower temperature range, and is more suitable for a low-temperature degradation environment.
The following are experimental steps for degrading high concentration herbicide under laboratory conditions for single/complex flora:
selecting strain, inoculating to 100mL1/10LB liquid medium, shake culturing at 4deg.C in shaker until OD 600 About 0.5, and the seed solution was inoculated in 1% of an inoculum size (0.33% inoculum size for each of three bacteria under a composite culture condition) into fresh 100mL1/10LB liquid medium (initial OD) 600 < 0.01), and 250mg/L of atrazine, acetochlor, butachlor, metolachlor, metribuzin, nicosulfuron, bensulfuron-methyl and fomesafen are added into the culture medium respectively; the cells were placed in a shaker at 4℃and cultured with shaking. Samples were taken every 6 hours. After centrifugation at 12000rpm for 2min, the change in herbicide content in the supernatant was detected by high performance liquid chromatography. Among them, the measurement method can be referred to example 1.
Table 2 degradation ability to eight herbicides in single/complex flora laboratory conditions
TABLE 3 degradation of Complex herbicides and improvement of soil ecological Functions by synthetic flora YKB and HKB in northeast Black soil
Example 3: (degradation of Complex herbicide and improvement of soil ecological Functions by synthetic flora YKB and HKB in northeast Black soil the inventors found from current research that the northeast Black soil region is more typical of five regionsThe types of residual herbicides in the soil of the zone were eight, including alachlor, acetochlor, butachlor, metolachlor, metribuzin, nicosulfuron, bensulfuron-methyl and fomesafen, but the concentration preferences were not the same, the herbicide residue conditions of the five zones were simulated according to the herbicide types and concentration preferences of the soil residues of the five zones, the soil function improvement experiment was performed, specifically, at about 10 ℃, two synthetic flora or composite microbial inoculum were added to the soil simulating the herbicide residue preferences of the five zones, respectively, and the soil was thoroughly stirred (initial biomass of microbial inoculum inoculated into the soil was 10% 4 cfu/g), mixing uniformly, standing for 35 days, sampling every 7 days, and detecting the content of different herbicides in soil.
In the course of the above experiment, the initial concentrations of herbicide in the soil simulating the residual conditions of herbicide in the above five regions were as follows (mg/kg):
liaoning Feilin Ling City: 92.5mg/kg of atrazine, 98.8mg/kg of acetochlor, 73.73mg/kg of butachlor, 49.81mg/kg of metolachlor, 19.98mg/kg of metribuzin, 17.48mg/kg of nicosulfuron, 11.25mg/kg of bensulfuron-methyl and 0.68mg/kg of fomesafen;
vintage Jilin province: 86.67mg/kg of atrazine, 94.17mg/kg of acetochlor, 34.85mg/kg of butachlor, 26.07mg/kg of metolachlor, 76mg/kg of metribuzin, 27.21mg/kg of nicosulfuron, 23.7mg/kg of bensulfuron-methyl and 0.35mg/kg of fomesafen;
harbin city, heilongjiang province: 66.5mg/kg of atrazine, 122.89mg/kg of acetochlor, 127.47mg/kg of butachlor, 45.66mg/kg of metolachlor, 26.17mg/kg of metribuzin, 12.82mg/kg of nicosulfuron, 5.757mg/kg of bensulfuron-methyl and 4.17mg/kg of fomesafen;
the city of the Heilongjiang province is seiulized: the alachlor is 85.8mg/kg, the acetochlor is 41.33mg/kg, the butachlor is 66.02mg/kg, the metolachlor is 17.31mg/kg, the metribuzin is 4.9mg/kg, the nicosulfuron is 7.44mg/kg, the bensulfuron-methyl is 3.96mg/kg and the fomesafen is 13.96mg/kg;
black river city of Heilongjiang province: 5.45mg/kg of atrazine, 17.16mg/kg of acetochlor, 7.78mg/kg of butachlor, 5.01mg/kg of metolachlor, 8.81mg/kg of metribuzin, 2.43mg/kg of nicosulfuron, 1.8mg/kg of bensulfuron-methyl and 84.4mg/kg of fomesafen.
From fig. 4 and fig. 5, in combination with table 4 and table 5, it can be seen that when the natural environment is about 10 ℃):
from the analysis of the complexity of removing the residual herbicide types in the soil, it can be known that the two composite bacterial agents can remove more than 90% of seven herbicides in the soil in the three regions of the simulated Heilongjiang province, the removal effect of the herbicide types on the soil is very considerable, the degradation rate of HBK is superior to that of YBK, and the microbial preparation containing HBK can be preferentially selected for the soil environment; in addition, for the two composite bacterial agents, the degradation rate of eight herbicides can reach over 70% basically for the simulated soil with eight residual herbicides in five areas, the degradation rate of the herbicides can be predicted for the complex herbicide environment of the soil, the degradation rate of HBK is better than that of YBK, and the microbial preparation containing HBK can be preferentially selected for the soil environment.
From the analysis of degradation rates of eight herbicides in the removed soil respectively, it can be known that the average degradation rate of the two composite microbial agents on eight herbicides can basically reach more than 80% in different soil environments, wherein the degradation rate of HBK is superior to that of YBK and can basically reach 90%, and the microbial preparation containing HBK can be preferentially selected in the complex soil environments with more herbicide types; in addition, the two composite bactericides have the best effect on the fomesafen herbicide in different soil environments, the degradation rate of the fomesafen herbicide can reach up to 97.52 percent, the degradation rate of sulfonylureas (nicosulfuron and bensulfuron-methyl) can reach up to 94 percent, the degradation rate of amides (acetochlor, butachlor and metolachlor) can reach up to 90 percent, and the degradation rate of triazines (atrazine and metribuzin) can reach up to 80 percent.
From the analysis of the two aspects, it can be known that the two composite microbial agents in the embodiment have considerable degradation rate of herbicide for the soil with a plurality of residual herbicides (the herbicide environment is complex), particularly under the low-temperature environment, relatively high degradation rate is kept for complex herbicide residues, the composite microbial agents are particularly suitable for being applied to northeast relatively low-temperature environments, and the application range is wider and the popularization significance is better compared with other microbial agents.
Referring to table 5, for eight herbicides remaining in the soil of two areas simulated therein, the degradation rate can reach 70% or more, and for seven herbicides remaining, the degradation rate can reach 75% or more, and for five herbicides remaining, the degradation rate can reach 80% or more, and for the soil where a plurality of herbicides remain (herbicide environment is complex), the degradation rate of the herbicide is very considerable, and particularly, high activity can be maintained in a low temperature environment (air temperature 10 ℃), so that the effect of improving the soil contaminated with the complex herbicide is foreseeable using the synthetic flora or the complex microbial inoculum referred to in the present application.
As can be seen from fig. 4, 5, 6 and 7, the synthetic flora in the invention has considerable degradation capability on eight herbicides in five areas, the degradation rate of the herbicides reaches over 63%, and most of the degradation rate can reach over 85%, wherein the degradation rate can reach over 90% for at least three herbicides remained in the soil in four simulated areas; five herbicides can be removed in one area, and the degradation rate reaches more than 90%; for at least six residual herbicides in the simulated four areas of soil, the degradation rate can reach more than 80%; the degradation rate of at least five residual herbicides in the simulated soil in five areas can reach more than 80 percent; the degradation rate of at least six residual herbicides in the simulated soil in five areas can reach more than 70 percent; referring to table 6, table 6 is a comparison of improvement of ecological functions in black soil (average value of improvement of each parameter after addition of two synthetic flora), wherein the control group is a soil sample, the restoration group is a soil sample added with a composite microbial inoculum, and as can be seen from table 6, the contents of beta-galactosidase, alpha-glucosidase, beta-glucosidase, urease, protease, phosphatase and dehydrogenase in the soil are increased, and malondialdehyde and catalase are reduced, so that the ecological functions of the soil are remarkably improved by adding the synthetic flora.
Table 4 degradation rates of two composite microbial agents for eight herbicides in five different areas within 35 days
Table 5 two composite microbial agents within 35 days, herbicide removal statistics (simultaneous degradation of different herbicide types and corresponding degradation rates)
TABLE 6 comparison of improvement of ecological functions in black soil
In conclusion, the two novel synthetic flora (or microbial inoculum) YKB and HKB designed by the invention have good degradation effects on eight kinds of composite herbicides currently existing in black soil in northeast areas under the condition of low temperature of about 10 ℃ (air environment), have no adverse effect on the environment, have higher universality and have better application prospects.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A low temperature degrading herbicide synthetic flora, comprising the following microbial strains:
bacillus [ ]Bacillus sp.) H1 and Comamonas spComamonas sp.) Any one of Y1 is respectively provided with a preservation number of CGMCC No.29207 and CGMCC No.29208;
pseudomonas spPseudomonas sp.) B1, the preservation number is CGMCC No.29205;
acinetobacter @Acinetobacter sp.) K1 with the preservation number of CGMCC No.29206.
2. The low temperature degrading herbicide synthesizing flora according to claim 1, wherein: the effective bacterial quantity ratio of the corresponding three strains contained in the synthetic flora is 1:1:1.
3. A composite microbial inoculant comprising the synthetic flora of claim 1.
4. A composite microbial agent according to claim 3, wherein: the compound microbial agent is obtained by mixing corresponding microbial strains in equal proportion.
5. Use of the synthetic flora of claim 1 or 2 or the complex inoculant of claim 3 or 4 for degrading at least one of alachlor, acetochlor, butachlor, metolachlor, metribuzin, nicosulfuron, bensulfuron-methyl and fomesafen herbicide.
6. Use of the synthetic flora of claim 1 or 2 or the complex inoculant of claim 3 or 4 for degrading at least one of residual atrazine, acetochlor, butachlor, metolachlor, metribuzin, nicosulfuron, bensulfuron-methyl and fomesafen herbicide in soil.
7. Use of the synthetic flora of claim 1 or 2 or the composite microbial inoculant of claim 3 or 4 for degrading at least one of alachlor, acetochlor, butachlor, metolachlor, metribuzin, nicosulfuron, bensulfuron-methyl and fomesafen herbicide under low temperature conditions, wherein the temperature of the low temperature conditions is in the range of-5-20 ℃.
8. Use of the synthetic flora of claim 1 or 2 or the composite microbial inoculant of claim 3 or 4 to degrade at least one of residual atrazine, acetochlor, butachlor, metolachlor, metribuzin, nicosulfuron, bensulfuron-methyl and fomesafen herbicide in soil under low temperature conditions, wherein the temperature of the low temperature conditions is 10 ℃.
9. Use of the synthetic flora according to claim 1 or 2 or the complex inoculant according to claim 3 or 4 for improving the ecological function of residual herbicide soil.
10. A method for improving the ecological function of residual composite herbicide soil is characterized by comprising the following steps: the method comprising applying to the soil an effective amount of the synthetic flora of claim 1 or 2 or the complex inoculant of claim 3 or 4.
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| CN114885952A (en) * | 2017-09-08 | 2022-08-12 | 马罗内生物创新公司 | Novel herbicidal compounds |
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