Herbicide halauxifen-methyl degrading strain and application thereof

Abstract

The invention provides a herbicide halauxifen-methyl degrading strain which is named Fusarium solani and preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.19365, and the preservation date is January 13, 2020. The herbicide halauxifen-methyl degrading strain has the advantages that the herbicide halauxifen-methyl degrading strain is named Fusarium solani, and the herbicide halauxifen-methyl degrading strain is named Fusarium solani; the nucleotide sequence of the ITS sequence of the fusarium solani is as shown in SEQ ID NO: 1 (sequence identifier number 1). The strain is used for degrading herbicide halauxifen-methyl. The strain disclosed by the invention provides an effective and available reference for subsequent research on biodegradation of halauxifen-methyl residues.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial strains, and in particular relates to a herbicide clofopyralid-methyl degrading strain and application thereof. Background Art

[0002] The use of herbicides has both advantages and disadvantages. Some herbicides do improve the efficiency of agricultural production, but they also bring about the problem of pesticide residues in the land. No matter how much medicine humans use on the land, it is likely to react to humans themselves along the food chain. Therefore, the safe use of all kinds of pesticides is increasingly closely related to the health of all of us. Therefore, the quality requirements of the world for herbicides now depend not only on the easy availability of raw materials, good weed control effects, and low costs, but also on whether it has irreversible harm to the crops planted and the surrounding animals, the physiological metabolism in animals and plants, and the ability of microorganisms in the soil to degrade it.

[0003] Microbial degradation is a natural ability, but natural microorganisms have a low degradation efficiency and a small degradation range for herbicides, and cannot be used as an effective means to target herbicide residues. Therefore, cultivating microorganisms with high efficiency in degrading herbicides is a new solution to target herbicide residues. In recent years, various microorganisms with high efficiency in degradation have gradually become the main solution to herbicide residues and have excellent development potential. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a herbicide clofopyralid-butyl degrading strain and its application in view of the above-mentioned deficiencies in the prior art, and the strain provides an effective and reliable reference for subsequent research on biodegradation of clofopyralid-butyl residues.

[0005] To solve the above technical problems, the technical solution adopted by the present invention is: a herbicide clofopyralid-butyl degrading strain, the herbicide clofopyralid-butyl degrading strain is named Fusarium solani, and is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No.: 19365, and the deposit date is January 13, 2020; the nucleotide sequence of the ITS sequence of Fusarium solani is shown in SEQ ID NO: 1.

[0006] The present invention also provides the use of the above-mentioned herbicide clofopyralid-methyl degrading strain, wherein the herbicide clofopyralid-methyl degrading strain is used to degrade the herbicide clofopyralid-methyl.

[0007] Compared with the prior art, the present invention has the following advantages: The herbicide clofopyralid-butyl degrading strain of the present invention provides an effective and reliable reference for subsequent research on biodegradation of clofopyralid-butyl residues.

[0008] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a colony morphology diagram obtained after purification in Example 1 of the present invention.

[0010] Figure 2 It is a standard curve diagram of the sample of Example 1 of the present invention.

[0011] Figure 3 This is a graph showing the degradation of clofopyralid by the strain of Example 1 of the present invention.

[0012] Figure 4 It is the 30 mg / L absorption peak of the standard product of Example 1 of the present invention.

[0013] Figure 5 It is the absorption peak of the control group in Example 1 of the present invention.

[0014] Figure 6 It is the absorption peak of the sample group of Example 1 of the present invention.

[0015] Figure 7 It is the residual concentration of clopidogrel after being treated with the strain of Example 1 of the present invention for 7 days.

[0016] Figure 8 This is a morphological identification diagram of the FLF1 strain in Example 1 of the present invention under an optical microscope.

[0017] Fig. 9 It is a phylogenetic tree diagram of the FLF1 strain of Example 1 of the present invention. DETAILED DESCRIPTION

[0018] Example 1 1.1 Experimental Materials The soil samples provided for this experiment were collected from the surface soil of rice fields in Xingwang Township, Nehe City, Heilongjiang Province. The collected soil was removed of impurities and set aside.

[0019] 1.2 Culture medium Basic inorganic salt medium: K2HPO4 1.79g, KH2PO4 0.45g, MgSO4·7H2O 0.2g, NaCl 0.4g, distilled water 1000mL, pH = 7.0. Solid medium: add agar powder 1.5-2.0%.

[0020] Inorganic salt + herbicide culture medium: After the basic inorganic salt culture medium is sterilized at 121℃ for 15 minutes, it is cooled to about 50℃ at room temperature, and clofopyralid technical is added as needed, and it is shaken well for later use.

[0021] Red Bengal culture medium: 5.0 g peptone, 10.0 g glucose, 1.0 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 19.0 g agar, 0.033 g red Bengal, 0.1 g chloramphenicol, 1000 mL distilled water, autoclave at 121°C for 15 min for use.

[0022] 1.3 Reagents and instruments 1.3.1 Main reagents Dichloromethane (Tianjin Guangfu, analytical grade); methanol (Shandong Yuwang, chromatographic grade); clofopyralid technical (currently Corteva, 91.4%); clofopyralid standard (Henan Alpha, 99% purity).

[0023] 1.4 Experimental methods 1.4.1 Separation and purification (1) Collect the soil and spread it out to air dry.

[0024] (2) Pour the air-dried soil into a mortar sterilized with ethanol, grind it into powder, and weigh 1 g to proceed with the following steps.

[0025] (3) Under sterile conditions, the soil sample was added into a flask containing 50 mg / L clofopyralid, with clofopyralid as the sole carbon and nitrogen source, and cultured on a constant temperature shaker at 30°C and 170 r / min.

[0026] (4) Take the culture and inoculate it into fresh basal inorganic salt medium every 3 days, and increase the content of clofopyralid in the medium by 50 mg / L. Continue acclimation and culture for 12 days until the final concentration of clofopyralid in the medium reaches 200 mg / L, and then the acclimation and culture is completed.

[0027] (5) The culture medium with a concentration of 100 mg / L of clofopyralid was diluted with sterile water for 10 -2 , 10 -4 , 10 -6 , 10 -8 Take 200 μL of the dilution containing bacteria, drip it onto the red Bengal medium at a uniform speed and spread it to make the bacteria evenly distributed in the medium. At the same time, make two parallel samples. Seal the culture dish with the sealing film prepared in advance, place it upside down in the incubator, and culture it at 28°C for 2-8 weeks.

[0028] (6) After culturing for a period of time, remove the culture dish from the incubator, pick out the mature strain and make three-zone lines on the Bengal red medium until a single colony appears. After purification, preserve the bacteria on a slant, cover it with a vent plug and refrigerate it for later use.

[0029] Test results: In the 50 mg / L shake flask, the growth of the bacteria could not be seen due to excessive turbidity. In the 150 mg / L and 200 mg / L shake flasks, almost no survival of the bacteria could be seen, indicating that the bacteria had no ability to degrade higher concentrations of clofopyralid. Therefore, the bacteria in the 100 mg / L shake flask were selected and diluted and spread on the LB plate. After one week of culture, obvious single colonies were produced, and white hyphae grew on the single colonies. The single colonies with relatively intact growth were picked for purification and culture to obtain the strain named FLF1.

[0030] Colony morphology: Figure 1 As shown, the purified strain was picked and inoculated on the Bengal red culture medium, and the resulting colony morphology was: white with hyphae, a bulge in the middle, neat edges, and no transparent circle around.

[0031] 1.4.2 Degradation ability of degrading bacteria After obtaining the purified strain, an inorganic salt bacterial suspension is made. Inoculate it in a basic inorganic salt culture medium, weigh and record before shaking, and shake at 30°C and 170r / min for 7 days. Set up a blank control CK, which is a liquid culture medium without bacteria, and shake it in the same way as the inoculated shake flask. Take a sample once after the time is up, weigh it according to the record to make up the water, then weigh and record it, and extract the culture fluid. Perform liquid chromatography analysis on the sample according to the optimal chromatographic conditions, record the peak area, calculate the residual concentration according to the standard curve of clofopyralid, and then calculate the degradation rate. After sampling the shake flask culture fluid, extract it. The extraction method steps are as follows: ① Add 0.25 g NaCl and 25 mL CH2Cl, shake for 1 min, and let stand to separate the layers.

[0032] ② Take out the upper aqueous phase and place it in another 10mL centrifuge tube, add 1mL of CH2Cl2, shake for 1min, and let it stand to separate the layers.

[0033] ③ Take the lower organic phase from the second centrifuge tube and place it in the corresponding centrifuge tube of the first centrifuge tube to combine the organic phases.

[0034] ④ Add 1 mL of CH2Cl2 to the second centrifuge tube, shake for 1 min, and let stand to separate the layers.

[0035] ⑤ Take the lower organic phase from the second centrifuge tube and place it in the corresponding centrifuge tube from the first centrifuge tube, combine the organic phases, add 0.2g of anhydrous Na2SO4 and shake.

[0036] ⑥ Remove a small amount of water from the organic phase, place the upper liquid in a 10mL volumetric flask, place the volumetric flask in a 50℃ constant temperature water bath, and evaporate CH2Cl2.

[0037] ⑦ Add methanol to make up to 10 mL, oscillate ultrasonically for 10 min, place in a 10 mL centrifuge tube, and place in a 4°C refrigerator for immediate testing.

[0038] After extraction, prepare for HPLC analysis under the following conditions: (1) Selection of detection wavelength: The ultraviolet spectrophotometer was used for spectral analysis, and the wavelength range of the largest absorption peak was 200-300 nm. The detection wavelength of the liquid chromatography was set to 200-260 nm, and the peak area was recorded. The peak area of ​​the same concentration standard at different wavelengths and the peak area of ​​different concentrations of standard at the same wavelength were compared. As shown in Table 1, the absorption peak of the standard sample was the largest at 210-240 nm, so 230 nm was selected as the analysis wavelength of the HPLC.

[0039] Table 1 Analysis of different wavelengths for different concentrations of standard products ; (2) Selection of mobile phase: Methanol: water = 3:1 and acetonitrile: phosphorus: water = 0.1%: 45:55 were selected for analysis in this experiment. The ideal liquid chromatography mobile phase solvent should have low viscosity, good compatibility with the monitor, easy to obtain pure products, and low toxicity. Therefore, the mobile phase was determined to be the former.

[0040] (3) Selection of flow rate: When the flow rate is 0-0.8 mL / min, the target peak cannot be retained for a long time and the impurity peak is not obvious; when the flow rate is 1.0 mL / min, the column pressure, retention time of the target peak and separation effect of its impurity peak are very good; when the flow rate is 1.0-1.5 mL / min, the column pressure is too high and the instrument is easily damaged. Therefore, 1.0 mL / min is selected as the flow rate.

[0041] Test results: (1) Drawing of standard curve The external standard method was used for quantification. Methanol was used as the solvent to prepare a 30 mg / L cloflupyridamole mother solution, which was then diluted to six standard concentration gradients of 1 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, and 25 mg / L. Chromatographic analysis was performed under the optimal chromatographic conditions. The standard curve was drawn based on the one-to-one correspondence between the measured peak area and the standard concentration, and the regression equation was established ( Figure 2 ).

[0042] (2) Determination of recovery rate In order to verify whether the pesticide formulation, extraction method and chromatographic conditions are reliable, this experiment used dichloromethane to extract clofopyralid in inorganic salt culture medium, diluted with methanol to 10mL, and the recovery of clofopyralid was determined under the optimal HPLC chromatographic conditions. The recovery rate was calculated by adding the following formula: Addition recovery rate (%) = (measured concentration / added concentration) × 100% The recovery rate reached 77.65%.

[0043] After shaking on the shaker for 7 days, the experimental group ( Figure 3 A) obtained bacterial suspension, control group ( Figure 3 B) No strain was generated, proving that the bacteria could grow using clofopyralid. The bacterial culture was then analyzed by HPLC ( Figure 4-6 ).

[0044] After 7 days of treatment with the strain, the residual concentration of 100 mg / L clofopyralid in the inorganic salt medium was Figure 7 As shown, the residual concentration in the control group was 77.65 mg / L, and the residual concentration in the experimental group was 42.59 mg / L. The calculated degradation rate of clofopyralid was 45.15%.

[0045] 1.4.3 Morphological identification under optical microscope Morphological identification method steps: (1) Place the specimen on a glass slide, add a drop of suspension solution, and cover with a coverslip.

[0046] (2) Leave it for a while or heat it slightly, that is, pass it quickly over the flame 2-3 times.

[0047] (3) Gently press the coverslip to expel bubbles and thin the specimen. Use filter paper to absorb the surrounding overflowing liquid and examine it under a microscope.

[0048] (4) When inspecting, block out strong light and first observe the presence of hyphae and spores under a low-power microscope. Then use a high-power microscope to observe the morphology, characteristics, position, size and arrangement of hyphae and spores.

[0049] Test results: After observation under an optical microscope, the morphology of the fungus was obtained. The fungus is a sickle-shaped fungus with hyphae ( Figure 8 ).

[0050] 1.4.4 ITS sequence sequencing (1) Genomic DNA extraction The procedure was performed according to the SK8259 (fungus) kit.

[0051] (2) PCR amplification (Table 2-4) Table 2 Universal primers for bacterial species identification ; Table 3 PCR amplification system ; Table 4 PCR reaction conditions ; (3) Gel electrophoresis 1% agarose gel electrophoresis, 150V, 100mA, 20min electrophoresis observation (4) Purification and recovery The desired DNA target band was cut from the electrophoresis band of the PCR product, and the PCR product was directly sequenced using PCR primers.

[0052] (5) ITS sequence analysis of FLA1 strain The above selected primers were used for sequence analysis to obtain the strain sequence results.

[0053] After sequencing, the strain was obtained as MK785401 in Genbank, with a length of 541 bp. The nucleotide sequence of the ITS sequence of the strain is shown in SEQ ID NO: 1. The phylogenetic tree is shown in Fig. 9 shown.

[0054] Through morphological observation of colony characteristics, strain FLF1 was white, opaque, round in shape, with a rough surface and frosted glass-like appearance. It was identified as a fungus by microscopic examination. After sequence alignment and phylogenetic tree construction, FLF1 had a 100% sequence homology with Fusarium solani. Combined with the morphological structure analysis of the colony, the degrading bacteria can be identified as Fusarium solani. This study has laid a theoretical foundation for microbial degradation of herbicides.

[0055] The strain is named Fusarium solani, and is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit address being: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No.: 19365, and the deposit date is January 13, 2020; the nucleotide sequence of the ITS sequence of the Fusarium solani is shown in SEQ ID NO: 1; the strain is used to degrade the herbicide clofopyralid.

[0056] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A herbicide clofopyralid-methyl degrading strain, characterized in that: The herbicide clofopyralid-butyl degrading strain was named Fusarium solani Fusarium solani , deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC No.: 19365 and the deposit date being January 13, 2020; the Fusarium solani Fusarium solani The nucleotide sequence of the ITS sequence is shown in SEQ ID NO:

1.

2. A use of the herbicide clofopyralid-degrading strain according to claim 1, characterized in that: The herbicide clofopyralid-methyl degrading strain is used for degrading the herbicide clofopyralid-methyl.

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