Composition for decomposing 2, 4-D butyl ester herbicide and application of composition

Abstract

The invention discloses a composition for decomposing a 2, 4-D butyl ester herbicide and application of the composition, and belongs to the technical field of pesticide degradation. In order to solve the problem of phytotoxicity of 2, 4-D butyl ester herbicides to schisandra chinensis, a mixed bacterium solution composed of bacillus amyloliquefaciens LC03, bacillus pumilus LC01, bacillus licheniformis LS04 and bacillus subtilis LS03 is used for degrading 2, 4-D butyl ester, and it is found that the mixed bacterium solution and a crude spore laccase solution generated by fermentation of the mixed bacterium solution can be used for degrading 2, 4-D butyl ester. Besides, the spore laccase crude enzyme liquid generated by fermentation of the mixed bacterial liquid is sprayed on the schisandra chinensis influenced by the 2, 4-D butyl ester, and the spore laccase crude enzyme liquid can be used for relieving the phytotoxicity of the 2, 4-D butyl ester herbicide on the schisandra chinensis, specifically, the yield and the chlorophyll content of the schisandra chinensis are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of pesticide degradation, and particularly relates to a composition for decomposing 2,4-D butyl ester herbicide and application thereof. Background Art

[0002] Schisandra chinensis (Trucz.) Braill., also known as Schisandra chinensis (Trucz.) Braill., is a plant of the Magnoliaceae family. Its fruit is used as medicine and is one of the most widely used important medicinal species. It has become one of the largest medicinal plants in Northeast China. Schisandra cultivation gardens are mostly adjacent to corn fields and soybean fields. With the continuous expansion of Schisandra cultivation areas and the widespread use of herbicides for field crops, Schisandra chinensis pesticide damage is common.

[0003] 2,4-D butyl ester is a phenoxyacetic acid type, hormone type, selective systemic herbicide that can be absorbed by the roots, stems, and leaves of plants. It has strong permeability and can easily enter the plant body. It kills the unearthed broadleaf weeds quickly. In addition, it is low in price, so it is widely used by grain farmers. However, this type of herbicide is highly volatile and can drift with wind or fog to sensitive crops several kilometers away from the application field. This drift can cause serious phytotoxicity to many broadleaf crops such as Schisandra chinensis, Asarum, and Dioscorea strychnifolia. The symptoms of phytotoxicity caused to Schisandra chinensis are withering and death of the growing point, dark green leaves, shrubs, failure to stretch vines, iron absorption disorders, flower buds drying up from the outside to the inside, browning and necrosis of mosaics and leaves, no pollen in stamens, dry pistil stigma, and failure to bear fruit normally, which in turn causes serious yield reduction and even death of the entire aboveground plant.

[0004] In addition to general field management, the methods reported in the literature to alleviate the damage caused by 2,4-D butyl ester mainly use spraying growth regulators such as gibberellins, auxin, and yuntaisu for reverse regulation, but the above methods are mainly used in economic crops such as cotton and grapes. There are few reports on the alleviation of drug damage to medicinal plants, especially the use of biological agents to alleviate the damage caused by 2,4-D butyl ester to Schisandra chinensis. Summary of the invention

[0005] In order to solve the phytotoxicity of 2,4-D butyl ester herbicide on Schisandra chinensis, the present invention uses a mixed bacterial liquid composed of Bacillus amyloliquefaciens LC03, Bacillus pumilus LC01, Bacillus licheniformis LS04 and Bacillus subtilis LS03 to degrade 2,4-D butyl ester, and it is found that the mixed bacterial liquid and the crude spore laccase enzyme liquid produced by the fermentation of the mixed bacterial liquid can be used to degrade 2,4-D butyl ester, and the crude spore laccase enzyme liquid produced by the fermentation of the mixed bacterial liquid can be used to alleviate the phytotoxicity of 2,4-D butyl ester herbicide on Schisandra chinensis.

[0006] In order to solve the above technical problems and obtain corresponding technical effects, the present invention provides the following technical solutions:

[0007] The first object of the present invention is to provide a composition for decomposing 2,4-D butyl ester herbicide, wherein the composition is composed of the following components: Bacillus amyloliquefaciens LC03, Bacillus pumilus LC01, Bacillus licheniformis LS04 and Bacillus subtilis LS03.

[0008] In one embodiment of the present invention, the concentrations of Bacillus amyloliquefaciens LC03, Bacillus pumilus LC01, Bacillus licheniformis LS04 and Bacillus subtilis LS03 in the composition are not less than 10 6 CFU / mL.

[0009] In one embodiment of the present invention, the composition is composed of the following ingredients: a concentration of 1×10 6 CFU / mL~5×10 6 CFU / mL of Bacillus amyloliquefaciens LC03, with a concentration of 1×10 6 CFU / mL~5×10 6 CFU / mL of Bacillus pumilus LC01, with a concentration of 1×10 6 CFU / mL~5×10 6 CFU / mL of Bacillus licheniformis LS04 and a concentration of 1×10 6 CFU / mL~5×10 6 CFU / mL of Bacillus subtilis LS03.

[0010] The second object of the present invention is to provide the use of the above composition in the degradation of 2,4-D butyl ester.

[0011] The third object of the present invention is to provide a method for preparing a crude enzyme solution of spore laccase using the above-mentioned composition, wherein the composition is inoculated into an LB liquid culture medium, cultured until the OD value reaches 1.5, to obtain a mixed bacterial solution, and then the mixed bacterial solution is inoculated into a spore-forming liquid culture medium, and the culture is stopped when more than 90% of empty spores are formed, and lysozyme is added for treatment to obtain a crude enzyme solution.

[0012] In one embodiment of the present invention, the composition of the spore-producing liquid culture medium is nutrient broth 8 g / L, KCl 1 g / L, MgSO4·7H2O 0.25 g / L, MnCl2·4H2O 0.002 g / L, CaCl2 0.5 mmol / L, and FeSO4 0.001 mmol / L.

[0013] The fourth object of the present invention is to provide a crude spore laccase solution prepared by the above method.

[0014] The fifth object of the present invention is to provide the use of the crude spore laccase solution in the degradation of 2,4-D butyl ester.

[0015] The sixth object of the present invention is to provide the use of the crude spore laccase solution in alleviating the phytotoxicity of 2,4-D butyl ester herbicide on Schisandra chinensis.

[0016] In one embodiment of the present invention, the mitigation of the phytotoxicity of Schisandra chinensis to 2,4-D butyl ester herbicide refers to increasing the yield and chlorophyll content of Schisandra chinensis.

[0017] Beneficial effects of the present invention:

[0018] The present invention uses a mixed bacterial liquid composed of Bacillus amyloliquefaciens LC03, Bacillus pumilus LC01, Bacillus licheniformis LS04 and Bacillus subtilis LS03 to degrade 2,4-D butyl ester, and it is found that the mixed bacterial liquid and the crude spore laccase enzyme liquid produced by fermentation of the mixed bacterial liquid can be used to degrade 2,4-D butyl ester. Under optimal reaction conditions, the degradation efficiency of the crude spore laccase enzyme liquid produced by fermentation of the mixed bacterial liquid for 2,4-D butyl ester can reach 90%.

[0019] In addition, the present invention uses the crude enzyme solution of spore laccase produced by fermentation of mixed bacterial liquid to spray Schisandra chinensis affected by 2,4-D butyl ester, and it is found that compared with the treatment without spraying the crude enzyme solution of spore laccase, the crude enzyme solution of spore laccase can increase the yield recovery rate of Schisandra chinensis by more than 20% and increase the chlorophyll content of Schisandra chinensis by more than 45%. The method for degrading the phytotoxicity of Schisandra chinensis provided by the present invention has the characteristics of environmental protection, high efficiency, safety, etc., and can be used to alleviate the phytotoxicity of Schisandra chinensis caused by 2,4-D butyl ester herbicide. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific implementation methods. The embodiments described in the present invention are not all embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The experimental methods used in the following embodiments are conventional methods unless otherwise specified, and the materials, reagents and instruments used are conventional materials, reagents and instruments in the field unless otherwise specified, and those skilled in the art can obtain them through commercial channels.

[0021] The strains used in the present invention are specifically as follows:

[0022] Bacillus amyloliquefaciens LC03, with a deposit number of CGMCC No. 4259, is disclosed in the invention patent with patent number ZL201010599756.4; Bacillus pumilus LC01, with a deposit number of CGMCC No. 4257, is disclosed in the invention patent with patent number ZL201010599461.7; Bacillus licheniformis LS04, with a deposit number of CGMCC No. 4263, is disclosed in the patent application with application number CN201010599481.4; Bacillus subtilis LS03, with a deposit number of CGMCC No. 4262, is disclosed in the patent application with application number CN201010599491.8.

[0023] Example 1: A composition for degrading 2,4-D butyl ester

[0024] The composition is composed of the following ingredients: Bacillus amyloliquefaciens LC03, Bacillus pumilus LC01, Bacillus licheniformis LS04 and Bacillus subtilis LS03.

[0025] The compounding method is as follows: Bacillus amyloliquefaciens LC03, Bacillus pumilus LC01, Bacillus licheniformis LS04 and Bacillus subtilis LS03 are inoculated into LB liquid culture medium respectively, and bacterial liquids containing Bacillus amyloliquefaciens LC03, Bacillus pumilus LC01, Bacillus licheniformis LS04 and Bacillus subtilis LS03 are obtained by culturing respectively, and the four bacterial liquids are mixed, and the concentrations of Bacillus amyloliquefaciens LC03, Bacillus pumilus LC01, Bacillus licheniformis LS04 and Bacillus subtilis LS03 in the mixed bacterial liquid are all 10 6 CFU / mL.

[0026] Example 2: Preparation of crude spore laccase solution

[0027] The formula of the spore-forming liquid culture medium is as follows: nutrient broth 8 g / L, KCl 1 g / L, MgSO4·7H2O 0.25 g / L, MnCl2·4H2O 0.002 g / L, adjusted to pH 7.0 and sterilized by high temperature and high pressure, and added with filtered sterilized 0.5 mmol / L CaCl2 and 0.001 mmol / L FeSO4.

[0028] The composition prepared in Example 1 was inoculated into LB liquid culture medium and cultured with shaking until the OD value reached 1.5 to obtain a mixed bacterial solution; the mixed bacterial solution was then inoculated into a spore-forming liquid culture medium with an inoculation amount of 1% of the volume of the spore-forming liquid culture medium, and cultured in a 20L microbial culture tank at 37°C with oxygen supply, stirring, and culturing for 36 hours, and the culture was stopped when more than 90% of empty spores were formed; lysozyme was added to the culture solution at a final concentration of 0.1 g / L and treated for 20 minutes to form a crude enzyme solution.

[0029] Example 3: Comparison of spore-producing laccase activity between composite bacterial consortium and single bacterial strain

[0030] According to the preparation method of the crude spore laccase solution in Example 2, crude enzyme solutions obtained by single strains of Bacillus amyloliquefaciens LC03, Bacillus pumilus LC01, Bacillus licheniformis LS04, and Bacillus subtilis LS03 through spore-forming liquid culture medium were prepared respectively, and according to the preparation method of the crude spore laccase solution in Example 2, crude spore laccase solution of mixed culture of four strains of Bacillus amyloliquefaciens LC03, Bacillus pumilus LC01, Bacillus licheniformis LS04 and Bacillus subtilis LS03 was prepared. The enzyme activity of laccase was determined by the guaiacol method (Liu Huan, Yuan Xingzhong, Peng Xin, et al. Study on laccase catalysis of guaiacol in reverse micelle system [J]. China Environmental Science, 2015, 35(02): 493-498.). The enzyme activity results are shown in Table 1 below. It can be seen that the spore laccase produced by mixed culture of the four bacteria has the highest activity.

[0031] Table 1 Comparison results of laccase activity

[0032]

[0033] Example 4: Method for decomposing 2,4-D butyl ester using the composition prepared in Example 1

[0034] The degradation of 2,4-D butyl ester by the composition prepared in Example 1 was carried out in a 3 mL system, the buffer was 50 mmol / L citric acid-phosphate buffer, the initial concentration of 2,4-D butyl ester was 5 mg / L, the pH value of the system was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0, respectively, the amount of the composition added in Example 1 was 1 mL, and the content of 2,4-D butyl ester in the reaction system with different pH was detected after reacting at 30°C for 24 hours, and the degradation rate of 2,4-D butyl ester was calculated to determine the effect of pH. The method for detecting the content of 2,4-D butyl ester is specifically referred to the following literature: Zhao Guiqin, Ju Zeliang, Chai Jikuan, Effect of herbicide 2,4-D butyl ester on safety and yield of hulled oats and residual dynamic analysis, Acta Prataculturae Sinica, 2018 27(06): 148-157. The calculation formula of the degradation rate is (initial concentration of 2,4-D butyl ester-final concentration of 2,4-D butyl ester) / initial concentration of 2,4-D butyl ester×100%. The results show that when the system pH is 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0, the degradation rates of 2,4-D butyl ester are 32.6%, 51.1%, 71.4%, 86.9%, 94.0% and 82.6%, respectively. Therefore, when the system pH is 8.0, the composition has the best degradation effect on 2,4-D butyl ester.

[0035] Example 5: Method for degrading 2,4-D butyl ester using the crude enzyme solution obtained in Example 2

[0036] The degradation of 2,4-D butyl ester by the crude enzyme solution obtained in Example 2 was carried out in a 3 mL system, the buffer was 50 mmol / L citric acid-phosphate buffer, the initial concentration of 2,4-D butyl ester was 5 mg / L, the pH value of the system was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0, respectively, the concentration of the crude enzyme solution was 25 U / L, and the content of 2,4-D butyl ester in the reaction system with different pH was detected after reacting at 30°C for 24 hours, and the degradation rate of 2,4-D butyl ester was calculated to determine the effect of pH. The results showed that when the system pH was 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0, the degradation rates of 2,4-D butyl ester were 23.4%, 53.8%, 74.3%, 80.7%, 85.2% and 76.1%, respectively. Therefore, when the system pH was 6.0-9.0, the crude enzyme solution had a better degradation effect on 2,4-D butyl ester, and when the system pH was 8.0, the crude enzyme solution had the best degradation effect on 2,4-D butyl ester.

[0037] Under the optimum pH condition, four different crude enzyme concentrations (10U / L, 25U / L, 50U / L and 100U / L) were set. After reacting at 30℃ for 24h, the content of 2,4-D butyl ester in the reaction system containing different concentrations of crude enzyme solution was detected, and the degradation rate of 2,4-D butyl ester was calculated to determine the effect of crude enzyme solution concentration. The results showed that when the crude enzyme solution concentration was 10U / L, 25U / L, 50U / L and 100U / L, the degradation rates of 2,4-D butyl ester were 56.7%, 83.6%, 82.9% and 80.1%, respectively. Therefore, when the crude enzyme solution concentration was 25U / L-100U / L, the crude enzyme solution had a better degradation effect on 2,4-D butyl ester, and when the crude enzyme solution concentration was 25U / L, the crude enzyme solution had the best degradation effect on 2,4-D butyl ester.

[0038] At the optimum pH value and crude enzyme concentration, the reaction temperatures were set at 15℃, 20℃, 30℃, 40℃ and 50℃, respectively. After 24 hours of reaction, the content of 2,4-D butyl ester corresponding to different reaction temperatures was detected, and the degradation rate of 2,4-D butyl ester was calculated to determine the effect of temperature. The results showed that when the reaction temperatures were 15℃, 20℃, 30℃, 40℃ and 50℃, the degradation rates of 2,4-D butyl ester were 59.4%, 63.8%, 75.9%, 85.6% and 79.7%, respectively. Therefore, when the reaction temperature was 30-50℃, the crude enzyme solution had a better degradation effect on 2,4-D butyl ester, and when the reaction temperature was 40℃, the crude enzyme solution had the best degradation effect on 2,4-D butyl ester, that is, the optimum reaction temperature was 40℃.

[0039] Subsequently, the degradation effect of the crude enzyme solution on 2,4-D butyl ester was tested under the conditions of optimal pH 8.0, crude enzyme solution concentration of 25U / L, and reaction temperature of 40°C. The results showed that the degradation rate of 2,4-D butyl ester increased with the extension of enzyme action time, reaching a degradation rate of 90% in about 24 hours.

[0040] Example 6: Crude enzyme solution improves chlorophyll content and yield of Schisandra chinensis damaged by 2,4-D butyl ester

[0041] Set up 3 processes:

[0042] Treatment 1: 2,4-D butyl ester + crude enzyme solution obtained in Example 2 (25 U / L);

[0043] Treatment 2: 2,4-D butyl ester + 10-fold dilution of the crude enzyme solution obtained in Example 2;

[0044] Treatment 3: 2,4-D butyl ester + spore-forming medium stock solution (obtained in Example 2).

[0045] Ten 10-year-old Schisandra chinensis plants were selected for each treatment, and three replicates were set. All treatments were sprayed with 0.045 mL / m 2 2,4-D butyl ester, 24 hours later, crude enzyme solution, 10 times dilution of crude enzyme solution and 10 ml of spore-forming medium solution were sprayed on the leaves of treatment 1, treatment 2 and treatment 3 respectively, and sprayed again after 7 days. After 15 days, the chlorophyll content of Schisandra chinensis leaves and the final Schisandra chinensis yield were recorded.

[0046] The method for determining the chlorophyll content is to select leaves that represent the general situation, wipe them clean, cut them into pieces, mix them, weigh 0.2g of each and put them into a mortar, add a small amount of quartz sand, CaCO3 and 2-3mL of 80% acetone solution by volume, grind them into a homogenate, add 5mL of 80% acetone solution by volume to continue grinding, transfer all of them into a 25mL brown volumetric flask, wash the mortar and pestle with 80% acetone solution by volume, and determine the absorbance value with an ultraviolet spectrophotometer at wavelengths of 663nm and 645nm, respectively. The leaves are repeated 5 times. The calculation formula is as follows:

[0047] Chlorophyll a:C a =12.7A 663 -2.69A 645

[0048] Chlorophyll b:C b =22.9A 645 -4.68A 663

[0049] The chlorophyll content of Schisandra chinensis leaves and the yield test results of Schisandra chinensis are shown in Table 2.

[0050] Table 2 Detection results of chlorophyll content of Schisandrae Chinensis leaves and yield of Schisandrae Chinensis

[0051]

[0052] Note: Different letters represent significant differences at the 0.05 level

[0053] It can be seen from the table that compared with treatment 3, the chlorophyll a content of treatment 1 and treatment 2 increased by 21.2% and 27.0%, the chlorophyll b content increased by 32% and 52%, and the yield increased by 19.9% ​​and 24.5%, respectively.

[0054] Although the present invention has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A composition for decomposing 2,4-D butyl ester herbicide, characterized in that: The composition is composed of the following ingredients: Bacillus amyloliquefaciens LC03, Bacillus pumilus LC01, Bacillus licheniformis LS04 and Bacillus subtilis LS03.

2. The composition according to claim 1, characterized in that The concentrations of Bacillus amyloliquefaciens LC03, Bacillus pumilus LC01, Bacillus licheniformis LS04 and Bacillus subtilis LS03 in the composition are not less than 10 6 CFU / mL.

3. The composition according to claim 2, characterized in that The composition is composed of the following ingredients: a concentration of 1×10 6 CFU / mL~5×10 6 CFU / mL of Bacillus amyloliquefaciens LC03, with a concentration of 1×10 6 CFU / mL~5×10 6 CFU / mL of Bacillus pumilus LC01, with a concentration of 1×10 6 CFU / mL~5×10 6 CFU / mL of Bacillus licheniformis LS04 and a concentration of 1×10 6 CFU / mL~5×10 6 CFU / mL of Bacillus subtilis LS03.

4. Use of the composition according to any one of claims 1 to 3 in the degradation of 2,4-D butyl ester.

5. A method for preparing a crude spore laccase solution using the composition according to any one of claims 1 to 3, characterized in that: The composition is inoculated into LB liquid culture medium and cultured until the OD value reaches 1.5 to obtain a mixed bacterial solution, which is then inoculated into a spore-forming liquid culture medium and cultured until more than 90% of empty spores are formed, and lysozyme is added to obtain a crude enzyme solution.

6. The use according to claim 5, characterized in that: The spore-producing liquid culture medium is composed of 8 g / L nutrient broth, 1 g / L KCl, 0.25 g / L MgSO4·7H2O, 0.002 g / L MnCl2·4H2O, 0.5 mmol / L CaCl2, and 0.001 mmol / L FeSO4.

7. The crude spore laccase solution prepared by the method according to any one of claims 5 or 6.

8. Use of the crude spore laccase solution according to claim 7 in the degradation of 2,4-D butyl ester.

9. Use of the crude spore laccase solution according to claim 7 in alleviating the phytotoxicity of 2,4-D butyl ester herbicide on Schisandra chinensis.

10. The use according to claim 9, characterized in that: The mitigation of the phytotoxicity of Schisandra chinensis to 2,4-D butyl ester herbicide refers to increasing the yield and chlorophyll content of Schisandra chinensis.

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