Strain for degrading quizalofop-p-ethyl and application

Abstract

The invention provides a bacterial strain for degrading quizalofop-p-ethyl and application of the bacterial strain. The technical problem that in the prior art, the effect of degrading quizalofop-p-ethyl by microorganisms is poor is solved. The invention provides a bacterial strain for degrading quizalofop-p-ethyl and application of the bacterial strain. The biological preservation number is CGMCC (China General Microbiological Culture Collection Center) No.31610. The strain is named as MJ-8; the MJ-8 bacterial strain belongs to the P.megatherium (P.megatherium). The MJ-8 strain can effectively degrade quizalofop-p-ethyl to generate low-toxicity or non-toxic products. And the MJ-8 strain can grow in a culture medium containing quizalofop-p-ethyl, which indicates that the MJ-8 strain can adapt to the existence of quizalofop-p-ethyl and can metabolize by using quizalofop-p-ethyl as a carbon source. Due to the capacity, the MJ-8 strain has huge application potential in the field of actual bioremediation, and secondary pollution can be avoided when the MJ-8 strain is used for bioremediation. In addition, the degradation rates of the MJ-8 strain on clodinafop-propargyl, haloxyfop-R-methyl, cyhalofop-butyl, fluazifop-p-butyl and benafop-p-ethyl all reach 90% or above, and a basis is provided for application of the MJ-8 strain in agricultural soil pollution remediation.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain for degrading quizalofop-ethyl and an application thereof. Background Art

[0002] Chemical herbicides play an important role in modern agriculture, especially chiral herbicides such as aryloxyphenoxypropionates (AOPPs), which have attracted widespread attention due to their high herbicidal activity. AOPP herbicides achieve weed control by inhibiting the activity of acetyl-CoA carboxylase (ACCase) and blocking the biosynthesis of weed fatty acids. Quizalofop-p-ethyl is one of the representatives of AOPP herbicides, which is mainly used to control grass weeds in broad-leaved crop fields. However, the use of quizalofop-p-ethyl poses a threat to the environment and human health. It is strictly controlled by many countries and is even listed as a possible human carcinogen. There are reports that quizalofop-p-ethyl may cause liver damage, and studies have found that it may cause obesity. In addition, quizalofop-p-ethyl has serious effects on the embryonic development and endocrine system of aquatic organisms such as zebrafish, and is also toxic to earthworms. Experimental studies have also shown that quizalofop-p-ethyl is cytotoxic and genotoxic, and can inhibit cell division and cause chromosomal aberrations. Other studies have shown that quizalofop-p-ethyl inhibits pollen germination and pollen tube growth.

[0003] In order to reduce the impact of quizalofop-p-ethyl and its metabolites on the ecological environment, it is crucial to study its degradation process in the environment. At present, the degradation methods of pesticide residues include photodegradation, plant degradation and microbial degradation. Among them, microbial degradation has received attention due to its key role in environmental remediation, especially in reducing the pesticide load in soil and water. Compared with physical and chemical methods, microbial degradation has the advantages of no secondary pollution, self-reproduction of microorganisms, and continuous degradation ability, which reduces the cost and labor of repeated additions. However, the efficiency of microbial degradation of quizalofop-p-ethyl may be affected by many factors, such as ambient temperature, humidity, competition from indigenous microorganisms, etc., which may limit its degradation efficiency in practical applications. In addition, the adaptability and colonization ability of efficient degrading bacteria screened or modified under laboratory conditions may be limited in the natural environment, making it difficult to achieve the expected degradation efficiency in contaminated soil. Therefore, although the microbial degradation method has obvious advantages, these challenges still need to be overcome in practical applications to improve the efficiency and effect of microbial degradation of quizalofop-p-ethyl. Summary of the invention

[0004] The present invention provides a strain for degrading quizalofop-p-ethyl and application thereof, aiming to solve the technical problem that the degradation effect of quizalofop-p-ethyl by microorganisms in the prior art is poor.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides a strain for degrading Quizalofop-P-ethyl, with a biological collection number of CGMCC No. 31610; the strain is named MJ-8; the MJ-8 strain belongs to Prioccidioides giganteus.

[0007] In a preferred solution, the conditions for the degradation of Quizalofop-P-ethyl by the MJ-8 strain are: pH 6-7, temperature 25-35° C., and inoculation amount 1%-9%.

[0008] Based on the above scheme, under the conditions, the metabolic activity and degradation ability of the MJ-8 strain reach the best, so that quizalofop-p-ethyl can be more effectively degraded and its residues in the environment can be reduced.

[0009] Preferably, the method for cultivating the MJ-8 strain is:

[0010] S1. Cultivate the strain: inoculate the strain MJ-8 into the culture medium, and culture it at a temperature of 28-32°C and a shaking speed of 150-170 rpm until the strain reaches the logarithmic growth phase;

[0011] S2. Collect strains: Centrifuge the cultured strain for 8-12 minutes, discard the supernatant, and wash the bacteria at least three times with MSM medium to obtain the MJ-8 strain.

[0012] Based on the above scheme, the control of temperature and shaker speed helps to maintain the oxygen supply and uniform distribution of nutrients required for strain growth, thereby ensuring the healthy growth and activity of the strain. The strain is cultured to the logarithmic growth phase because the bacteria in the logarithmic growth phase have strong metabolic activity and are suitable for subsequent degradation tests. Centrifugal washing removes unnecessary impurities and culture medium components in the culture medium to obtain a pure MJ-8 strain.

[0013] In a preferred embodiment, in step S1, the culture medium is NB culture medium.

[0014] Based on the above scheme, NB medium can provide nitrogen source, B vitamins, amino acids and other growth factors for the growth of MJ-8 strain, and is a medium suitable for bacterial growth.

[0015] In a second aspect, the present invention provides use of the MJ-8 strain described in the first aspect in the preparation of a degradable herbicide Quizalofop-P-ethyl.

[0016] A preferred embodiment is the use of the strain in degrading the quizalofop-ethyl herbicide in soil.

[0017] Based on the above scheme, the MJ-8 strain can effectively degrade quizalofop-p-ethyl in the soil and reduce its residue in the environment. The application of the MJ-8 strain in degrading quizalofop-p-ethyl herbicide in the soil not only helps to control environmental pollution, but also promotes the healthy development of soil microorganisms.

[0018] The beneficial effects of the present invention are:

[0019] The present invention provides a strain and application for degrading quizalofop-p-butyl. It has been proved by experiments that the MJ-8 strain can effectively degrade quizalofop-p-butyl and generate products with low toxicity or no toxicity. And the MJ-8 strain can grow in a culture medium containing quizalofop-p-butyl, which shows that it can adapt to the presence of quizalofop-p-butyl and can metabolize quizalofop-p-butyl using quizalofop-p-butyl as a carbon source. This ability makes the MJ-8 strain have great application potential in the actual field of bioremediation. Compared with traditional physical and chemical degradation methods, the use of the MJ-8 strain for bioremediation can avoid the generation of secondary pollution and provide a more environmentally friendly solution. In addition, the MJ-8 strain has a certain degradation ability for other AOPP herbicides. It has been proved by experiments that the MJ-8 strain has a high degradation activity for clodinafop-butyl, high-efficiency fluazifop-butyl, cyhalofop-butyl, fluazifop-p-butyl and diclofenac, and the degradation rate is more than 90%, and it also has a certain degradation effect on fenoxaprop-p-butyl and oxadiazon, which provides a basis for the application of the MJ-8 strain in agricultural soil pollution remediation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 It is the degradation of quizalofop-p-ethyl during the growth of strain MJ-8.

[0022] Figure 2 It is the degradation ability of strain MJ-8 on quizalofop-p-ethyl under different culture conditions.

[0023] Figure 3 This is the colony morphology of strain MJ-8.

[0024] Figure 4 This is an electron micrograph of strain MJ-8. DETAILED DESCRIPTION

[0025] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0026] Embodiment 1:

[0027] This embodiment provides a strain for degrading quizalofop-p-ethyl, the biological deposit number is CGMCC No. 31610; the deposit unit is the General Microbiology Center of China Microbiological Culture Collection Administration; the deposit date is August 6, 2024; the classification name is Priestia megaterium; the strain is named MJ-8; the MJ-8 strain belongs to Priestia megaterium.

[0028] The conditions for the degradation of Quizalofop-P-ethyl by the MJ-8 strain are: pH 6-7, temperature 25-35°C, and inoculation amount of at least 1%-9%.

[0029] Specifically, strain MJ-8 has a high degradation efficiency under weakly acidic and neutral conditions. The optimum temperature for strain MJ-8 to degrade quizalofop-p-ethyl is 30°C. The inoculation amount ranges from 1% to 9%. The degradation rate gradually increases with the increase of inoculation amount and the extension of time. However, when the inoculation amount is too high, the strain grows rapidly, which may lead to insufficient substrate for growth and metabolism. Experiments have shown that the most appropriate ratio of the concentration of MJ-8 strain to the concentration of quizalofop-p-ethyl is maintained between 1:10 and 1:9.

[0030] The cultivation method of the MJ-8 strain is:

[0031] S1. Cultivate the strain: inoculate the strain MJ-8 into the culture medium, and culture it at a temperature of 28-32°C and a shaking speed of 150-170 rpm until the strain reaches the logarithmic growth phase;

[0032] S2. Collect strains: Centrifuge the cultured strain for 8-12 minutes, discard the supernatant, and wash the bacteria at least three times with MSM medium to obtain the MJ-8 strain.

[0033] Specifically, S1. Cultivating strains: First, inoculate strain MJ-8 into NB medium and culture at a temperature of 30° C. and a shaking speed of 160 rpm until the strain reaches a logarithmic growth phase;

[0034] S2. Collect the strains: centrifuge the cultured strains at 5000 rpm for 10 minutes, and then discard the supernatant; wash the bacteria three times with fresh MSM medium;

[0035] Next, the OD600 of the bacterial suspension was adjusted to 1.0 to standardize the bacterial concentration; 4-6 parts by mass of the bacterial suspension were inoculated into 100 parts by mass of MSM medium containing 18-20 mg / L quizalofop-p-ethyl, and the reaction was carried out at 30° C. and 160 rpm for 7 days, and the degradation of quizalofop-p-ethyl was monitored every 12 hours by a UV spectrophotometer.

[0036] Embodiment 2:

[0037] This example provides the use of the MJ-8 strain described in Example 1 in the preparation of a degradable herbicide Quizalofop-P-ethyl.

[0038] The MJ-8 strain is used for degrading the quizalofop-ethyl herbicide in soil.

[0039] Specifically, the MJ-8 strain can be used to degrade the residues of the herbicide quizalofop-p-ethyl, reduce pesticide pollution in farmland soil and water, and can also be used as a bioremediation agent to treat soil and water contaminated by quizalofop-p-ethyl, degrade organic pollutants in the environment through its metabolic activities, and improve environmental quality.

[0040] The present invention will be further explained below in conjunction with experiments:

[0041] 1. Materials and methods

[0042] 1.1 Chemicals and culture media

[0043] Quizalofop-p-ethyl (purity 94.4%), cyhalofop-butyl (purity 96.44%), clodinafop-butyl (purity 95%), fluazifop-butyl (purity 99.1%), oxadiazolamide (purity 97.8%), oxadiazol-butyl (purity 98%), fluazifop-p-ethyl (purity 97%), diclofenac (purity 96%) were purchased from Hunan Xinchangshan Agricultural Development Co., Ltd. All pesticide technicals were dissolved in methanol (HPLC grade) to prepare stock solutions (1×10 4 mg / L) and sterilized using a filter with a pore size of 0.22 μm.

[0044] In this study, the bacteria were cultured using beef extract peptone broth (NB), Luria-Bertani (LB) medium and mineral salts medium (MSM).

[0045] 1.2 Isolation and identification of bacteria that degrade the herbicide Quizalofop-P-ethyl

[0046] The test samples were collected from the soil of a cotton field in Baoguo Village, Canggang Town, Hanshou County, Changde City, Hunan Province (N: 28°57′6", E: 111°52′46"), where quizalofop-ethyl is used all year round. The soil samples were added to MSM (basal salt medium) containing 20 mg / L quizalofop-ethyl. After incubation at 30°C and 160rpm for 7 days, the enrichment solution was diluted and spread on an MSM agar plate added with 20 mg / L quizalofop-ethyl. After culturing for 5 days, colonies were picked and streaked on the MSM plate until a single colony was obtained. The obtained strain was inoculated into an MSM medium containing 20 mg / L quizalofop-ethyl and cultured for 7 days, and its degradation ability was detected by HPLC. The strain with the highest degradation ability was selected and named MJ-8.

[0047] 1.3 Chemical analysis

[0048] The strain MJ-8 was inoculated into MSM medium for cultivation, and the blank control was used without inoculation. An equal volume of acetonitrile (HPLC) was added to the culture medium and centrifuged at 10000 rpm for 5 min. The supernatant was taken, the solution was filtered through a 0.22 μm membrane, and then analyzed by a high-performance liquid chromatograph (Agilent Technologies, USA) equipped with a C18 column (AthenaC18-WP, 5 μm, 4.6×250 mm). A mobile mobile phase consisting of acetonitrile: water (85:15 v / v) was used, the flow rate was 1.0 mL / min, the injection volume was 20 μL, the column temperature was maintained at 25 °C, and the UV detector was set at 234 nm. The concentration of quizalofop-ethyl was determined based on the peak area of ​​the standard curve. To detect the metabolic degradation products of quizalofop-ethyl, samples were obtained by the above method and analyzed by liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (HPLC-QTOFMS). Gradient elution was used, and MS analysis was operated with an electrospray ionization source in positive polarity mode.

[0049] 1.4 Investigating the effects of cell growth and culture conditions on the degradation of the herbicide quizalofop-p-ethyl by the MJ-8 strain

[0050] (1) Strain culture: The MJ-8 strain was inoculated into NB medium and cultured at 30°C and 160 rpm in a shaking incubator until the logarithmic growth phase.

[0051] (2) Collection and washing of bacterial cells: The cultured strains were centrifuged at 5000 rpm for 10 minutes, the supernatant was discarded, and the cells were washed three times with fresh MSM medium. The OD value of the bacterial suspension was calculated. 600 Adjusted to 1.0.

[0052] (3) Inoculation and incubation: The bacterial suspension was inoculated into 20 mL of MSM medium containing 20 mg / L quizalofop-p-ethyl at a ratio of 5% (v / v) and incubated at 30° C. and 160 rpm for 7 days.

[0053] (4) Growth monitoring: Use an ultraviolet spectrophotometer to monitor the bacterial growth every 12 hours, and set up samples without inoculation of strains as a control group.

[0054] (5) Optimization of culture conditions: The strain MJ-8 was inoculated in MSM medium containing 20 mg / L quizalofop-p-ethyl. The effects of different temperatures (20-40°C), medium pH (5-9), inoculation amount (1-9%, v / v) and initial concentration of quizalofop-p-ethyl (20-100 mg / L) on the degradation effect were investigated.

[0055] At the same time, the present invention also explores the degradation of other members of AOPP (cyhalofop-butyl, clodinafop-butyl, fluazifop-butyl, dimethoate, fluazifop-butyl, fenoxaprop-butyl and oxadiazine) by strain MJ-8. The residue was determined by the method described in Section 1.3.

[0056] 2. Results and Analysis

[0057] See also Figure 3 and Figure 4 , Figure 3 The growth of the MJ-8 strain on the plate is shown, in which the colonies are clearly visible and evenly distributed, indicating that the MJ-8 strain has vigorous growth vitality and good reproduction ability. Figure 4 The microstructure of the MJ-8 strain was revealed by electron microscopy images. In the high-resolution electron microscopy images, it can be clearly seen that the cell morphology of the strain is intact, and the cell wall and cell membrane structure show good integrity and stability.

[0058] Degradation dynamics of quizalofop-p-ethyl by strain MJ-8 in MSM liquid medium. Figure 1 Under the culture conditions of pH 7.0 and 30°C, the MJ-8 strain was inoculated into MSM medium containing 20 mg / L quizalofop-p-ethyl. After 96 hours, the cell concentration reached the maximum value. Compared with the control group, the residual rate of quizalofop-p-ethyl gradually decreased with the gradual increase of cell concentration. The growth curve of the MJ-8 strain was positively correlated with the degradation rate of quizalofop-p-ethyl.

[0059] In order to evaluate the potential of strain MJ-8 to degrade the herbicide quizalofop-p-ethyl, the effects of different culture conditions on the degradation effect were investigated. Figure 2 These culture conditions include pH level, culture temperature, inoculation amount and substrate concentration. The test results show that pH level has a significant effect on the degradation of quizalofop-p-ethyl by MJ-8 strain. Figure 2A, Under the conditions of pH 6-7, the degradation rates of quizalofop-p-ethyl reached 83.56% and 82.55% after 120 hours, respectively. However, under the conditions of pH 5, 8, and 9, the degradation rates of quizalofop-p-ethyl dropped to 49.25%, 62.74%, and 53.36% after 120 hours, respectively, indicating that lower or higher pH values ​​would seriously hinder the growth and degradation efficiency of the MJ-8 strain.

[0060] In addition, the culture temperature is also a key factor affecting the degradation performance of Quizalofop-Ph-ethyl. Figure 2 B. The test shows that the optimum temperature for the degradation of Quizalofop-Ph-ethyl by the MJ-8 strain is 30°C, while the degradation efficiency is low under low temperature conditions. This is because low temperature limits the growth of the strain, thus affecting the degradation efficiency. When the temperature is in the range of 35-40°C, the MJ-8 strain exhibits a higher degradation ability as the culture time increases. This is because the MJ-8 strain is a Bacillus, and its bacteria and the degradation enzymes it produces are more resistant to high temperatures, so the degradation efficiency is not greatly affected at higher temperatures.

[0061] Inoculum size is also an important factor affecting the degradation rate of Quizalofop-Ph-ethyl. Figure 2 C. In the experiment, the inoculation amount ranged from 1% to 9%. The results showed that as the inoculation amount increased, the degradation rate also gradually increased. The appropriate inoculation amount is conducive to the degradation of quizalofop-p-ethyl by microorganisms. Too little inoculation amount will lead to slow growth of the strain and affect cell survival rate. When the inoculation amount is too large, the strain will grow rapidly, which may lead to insufficient substrates for growth and metabolism. Figure 2 D. The substrate concentration also affects the degradation efficiency of Quizalofop-PhD. As the concentration of Quizalofop-PhD increases, its degradation rate gradually decreases, and the degradation rate also decreases. This shows that Quizalofop-PhD has a certain toxicity. The MJ-8 strain can effectively degrade Quizalofop-PhD within a certain range, but once the concentration of Quizalofop-PhD exceeds a certain limit, it will seriously harm the growth of the strain, thereby affecting the degradation efficiency.

[0062] In addition, see Figure 2 E, MJ-8 strain also showed high catalytic activity against other AOPP herbicides. Among the 7 AOPP herbicides tested, clodinafop-butyl was completely degraded, while the degradation rates of fluazifop-butyl, cyhalofop-butyl, fluazifop-butyl and diclofop-butyl were 99.63%, 91.71%, 99.6% and 99.99%, respectively. The degradation rates of fluazifop-butyl and oxadiazon were lower, at 46.99% and 28.13%, respectively.

[0063] 3. Test conclusion

[0064] This experiment investigated the degradation efficiency of Quizalofop-Ph-ethyl by the MJ-8 strain under different culture conditions. The following are the main findings and conclusions of the experiment:

[0065] (1) Strain growth and quizalofop-p-ethyl degradation: The MJ-8 strain has a good degradation ability for quizalofop-p-ethyl. Under the conditions of pH 7.0 and 30°C, the strain growth was positively correlated with the degradation of quizalofop-p-ethyl. The cell concentration reached the maximum after 96 hours, and the residual rate of quizalofop-p-ethyl was significantly reduced.

[0066] (2) Effect of culture conditions on degradation efficiency: The degradation efficiency of the MJ-8 strain was the highest under the conditions of pH 6 and 7, while the degradation efficiency decreased significantly at pH 5, 8 and 9. 30°C is the optimum temperature for the degradation of quizalofop-p-ethyl by the MJ-8 strain, and low temperature conditions limit the growth and degradation efficiency of the strain. Increasing the inoculation amount can improve the degradation efficiency of quizalofop-p-ethyl by the MJ-8 strain, but if the inoculation amount is too large, the growth and degradation ability of the strain may be limited due to insufficient substrate supply. At the same time, with the increase of quizalofop-p-ethyl concentration, both the degradation rate and degradation rate were observed to decrease. Therefore, in order to ensure the effectiveness of the degradation process, the inoculation amount needs to be adjusted according to the concentration of quizalofop-p-ethyl, and the ratio of the strain concentration to the quizalofop-p-ethyl concentration should be kept between 1:9 and 1:10. This ensures that the strain has sufficient substrate for degradation while maintaining a high degradation activity, thereby achieving more effective biodegradation effects at different quizalofop-p-ethyl concentrations.

[0067] (3) Degradation ability of other AOPP herbicides: The MJ-8 strain showed high degradation activity against clodinafop-butyl, fluazifop-butyl, cyhalofop-butyl, fluazifop-butyl and diclofop-butyl, with degradation rates exceeding 90%. It also had a certain degradation effect on fenoxaprop-butyl and oxadiazon, which indicated that the MJ-8 strain had a certain degradation ability against AOPP herbicides.

[0068] (4) Environmental remediation potential: The MJ-8 strain has a high degradation efficiency of quizalofop-p-ethyl under weakly acidic and neutral conditions, indicating that it has application potential in actual environmental remediation. The strain's temperature tolerance and ability to degrade quizalofop-p-ethyl make it a promising bioremediation tool.

[0069] In summary, the MJ-8 strain can effectively degrade the herbicide quizalofop-p-ethyl and has a certain degradation ability for other AOPP herbicides. These results provide a basis for the application of the MJ-8 strain in the remediation of agricultural soil and water pollution.

[0070] The present invention is not limited to the above-mentioned optional implementation modes. Anyone can derive other various forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, all technical solutions that fall within the scope defined by the claims of the present invention fall within the protection scope of the present invention.

Claims

1. A strain for degrading Quizalofop-P-ethyl, characterized in that: The biological preservation number is CGMCC No.31610; the strain is named MJ-8; the MJ-8 strain belongs to Priesteria gigantea.

2. The strain for degrading Quizalofop-ethyl according to claim 1, characterized in that: The conditions for the degradation of quizalofop-p-ethyl by the MJ-8 strain are: pH 6-7, temperature 25-35°C, and inoculation amount 1%-9%.

3. The strain for degrading Quizalofop-ethyl according to claim 1, characterized in that: The cultivation method of the MJ-8 strain is: S1. Cultivate the strain: inoculate the strain MJ-8 into the culture medium, and culture it at a temperature of 28-32°C and a shaking speed of 150-170 rpm until the strain reaches the logarithmic growth phase; S2. Collect strains: Centrifuge the cultured strain for 8-12 minutes, discard the supernatant, and wash the bacteria at least three times with MSM medium to obtain the MJ-8 strain.

4. The strain for degrading Quizalofop-ethyl according to claim 3, characterized in that: In step S1, the culture medium is NB culture medium.

5. An application of the MJ-8 strain according to any one of claims 1 to 4, characterized in that: The MJ-8 strain is used in the preparation of a quizalofop-p-ethyl herbicide for degradation.

6. The use of the strain according to claim 5, characterized in that: The MJ-8 strain is used in degrading the quizalofop-ethyl herbicide in soil and water.

Images