Quizalofop-p-ethyl degrading strain and application thereof

Abstract

The invention provides a quizalofop-p-ethyl degrading strain Aspergillus fumigatus, and the strain is used for degrading the quizalofop-p-ethyl. The Aspergillus fumigatus provided by the invention has the function of degrading the residual quizalofop-p-ethyl in soil, and the degradation rate of the Aspergillus fumigatus to the quizalofop-p-ethyl with the initial concentration of 100 mg / L within 7 days is 16.07%.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial applications, and specifically relates to a quizalofop-p-ethyl degrading strain and its application. Background Art

[0002] Quizalofop-p-ethyl (Q) is an aryloxyphenoxypropionate herbicide with a relatively high application rate. Due to its high selectivity between gramineous weeds and broad-leaved crops, it is often applied to broad-leaved crops such as soybeans, beets, and rapeseed. Its mechanism of action is as follows: After application, it enters the weed tissue and is hydrolyzed into chiral and biologically active quizalofop-p-ethyl acid, resulting in a decrease in the synthesis of acetyl-CoA carboxylase in plants. After being absorbed through the leaf surface and moving within the tissue, it accumulates in the meristem, inhibits the synthesis and metabolism process of fatty acids, destroys the membrane lipid cell membrane system, affects the cell growth and division process, causes physiological dysfunction of the meristem, and ultimately leads to the death of weeds. The national food safety standard of China, GB2763-2019, stipulates that the maximum residue limit of quizalofop-p-ethyl in soybeans is 0.1 mg•kg -1 . Many reports show that the residue of quizalofop-p-ethyl will have a serious impact on animals and plants. How to effectively reduce the residue after the application of quizalofop-p-ethyl has become an urgent problem to be solved. In recent years, it has been found that microorganisms such as bacteria, fungi, and actinomycetes can degrade residual pesticides in the soil. The combined use of multiple highly efficient degrading microorganisms can effectively improve the degradation efficiency of quizalofop-p-ethyl.

[0003] As the negative impact of herbicide residues on the environment and organisms has been increasingly emphasized, the degradation of pesticide residues has become an urgent problem to be solved. Using microorganisms to degrade chemical pesticide residues in the environment is an economical and effective method. Currently, strains of various genera have been isolated and proven to be able to degrade the residual quizalofop-p-ethyl in the soil, such as Methylobacterium extorquens, Bacillus subtilis, Rhodococcus, and Ochrobactrum. Most of the reported quizalofop-p-ethyl degrading bacteria are bacteria, and there are no reports on fungi that degrade quizalofop-p-ethyl. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a quizalofop-p-ethyl degrading strain and its application in view of the deficiencies of the above-mentioned prior art. This Aspergillus fumigatus has the function of degrading residual quizalofop-p-ethyl in the soil.

[0005] To solve the above technical problem, the technical solution adopted by the present invention is: A quizalofop-p-ethyl degrading strain, and the quizalofop-p-ethyl degrading strain is Aspergillus fumigatus.

[0006] The present invention also provides the application of the above-mentioned quizalofop-p-ethyl degrading strain, and the Aspergillus fumigatus is used for degrading quizalofop-p-ethyl.

[0007] Preferably, the degradation rate of the Aspergillus fumigatus to quizalofop-p-ethyl with an initial concentration of 100 mg / L within 7 days is 16.07%.

[0008] The present invention has the following advantages compared with the prior art: The Aspergillus fumigatus of the present invention has the function of degrading the residual quizalofop-p-ethyl in the soil, and the degradation rate of the strain to quizalofop-p-ethyl with an initial concentration of 100 mg / L within 7 days is 16.07%.

[0009] The present invention will be further described in detail below with reference to the drawings and embodiments. Description of the Drawings

[0010] Figure 1 It is a diagram showing the growth of mycelium at the bottom of each culture flask in Example 1 of the present invention.

[0011] Figure 2 It is the residual concentration of quizalofop-p-ethyl after 7 days of treatment with each primary screening strain in Example 1 of the present invention.

[0012] Figure 3 It is a diagram showing the cell morphology of QF2 under an optical microscope in Example 1 of the present invention.

[0013] Figure 4 It is a diagram showing the colony morphology of QF2 on an RBA medium in Example 1 of the present invention.

[0014] Figure 5 It is the result of multiple sequence alignment of the ITS sequence of the QF2 strain in Example 1 of the present invention.

[0015] Figure 6 It is the phylogenetic tree of the QF2 strain in Example 1 of the present invention. Detailed Embodiments

[0016] Example 1 For the quizalofop-p-ethyl degrading strain in this example, the quizalofop-p-ethyl degrading strain is Aspergillus fumigatus, and this quizalofop-p-ethyl degrading strain is used for degrading quizalofop-p-ethyl; the degradation rate of the Aspergillus fumigatus to quizalofop-p-ethyl with an initial concentration of 100 mg / L within 7 days is 16.07%.

[0017] The following are the isolation, identification of this strain and the test for degrading quizalofop-p-ethyl.

[0018] 1. Materials and Methods 1.1 Test Time and Location The test was carried out in the Key Laboratory of Cold Region Ecological Restoration and Resource Utilization in Heilongjiang Province in the second half of 2020.

[0019] 1.2 Test Materials, Reagents and Culture Media 1.2.1 Soil Samples: Soil from a paddy field in Harbin where quizalofop-p-ethyl has been applied for a long time 1.2.2 Reagents: 94% quizalofop-p-ethyl technical 1.2.3 Culture Media Basic Inorganic Salt Medium (MM): K2HPO4 1.79 g, KH2PO4 0.45 g, MgSO4·7H2O 0.2 g, NaCl 0.4 g, deionized water 1 L (15 - 20 g of agar powder is added for solid medium), adjust the pH to 7.0, sterilize at 0.1 Mpa and 121 °C by high-temperature and high-pressure moist heat for 20 min.

[0020] Quizalofop-p-ethyl Inorganic Salt Medium (MM + Q): After the MM sterilized by high-temperature and high-pressure moist heat is cooled to about 45 °C at room temperature, add a reasonable concentration of 94% quizalofop-p-ethyl technical dissolved in absolute ethanol (the volume of absolute ethanol is 1 - 5% of the volume of the medium) using a 0.22 μm filter membrane, and shake well.

[0021] Rose Bengal Medium (RBA): Rose bengal agar powder 35 g, agar powder 10 g, deionized water 1 L, sterilize at 0.1 Mpa and 121 °C by high-temperature and high-pressure moist heat for 20 min.

[0022] 1.3 Chromatographic Conditions Ultra-high performance liquid chromatograph: Waters ACQUITY UPLC H-Class, chromatographic column: ACQUITY UPLC HSST3 1.8 μm (3.1×100 mm), mobile phase: methanol: water = 80:20, flow rate: 1 mL / min, column temperature: 40 °C, injection volume: 3 μL, detection wavelength: 230 nm.

[0023] 1.4 Test Methods 1.4.1 Primary Screening of Quizalofop-p-ethyl Degrading Bacteria Refer to the enrichment and domestication of soil samples using the enrichment culture method: Weigh 1.0 g of soil samples into 50 mL of 50 mg / L quizalofop-p-ethyl inorganic salt medium, culture at 30 °C and 150 r / min with constant rotation for 3 d, then take 1 mL of the culture solution and inoculate it into 100 mg / L quizalofop-p-ethyl inorganic salt medium. After repeating the above operation, inoculate it into 150 mg / L and 200 mg / L quizalofop-p-ethyl inorganic salt medium. Gradient dilute the 10 mg / L MM + Q culture solution to 10 -5Five gradients were inoculated on the solid medium of MM+Q at 100 mg / L. After culturing at a constant temperature of 30 °C for one week, the fungal colonies growing on it were picked and purified on RBA to obtain the primary screening strains.

[0024] 1.4.2 Quantitative analysis of the degradation ability of quizalofop-p-ethyl-degrading strains The strains obtained from the primary screening were respectively inoculated into the liquid medium of MM+Q at 100 mg / L. Three replicate experimental groups were set for each strain. After weighing, they were cultured at a constant temperature of 30 °C and 150 r / min for 7 d. The Erlenmeyer flasks were taken out and replenished with water to the mass before cultivation. 1 mL of the culture solution was taken out from each flask, extracted and fixed to 10 mL with chromatographic methanol. The mother liquor of quizalofop-p-ethyl was prepared with chromatographic methanol and diluted into a quizalofop-p-ethyl standard series: 0.15, 3, 6, 9, 12, 15 mg / L. It was analyzed by ultra-high performance liquid chromatography together with the extracted samples. The standard curve of quizalofop-p-ethyl was drawn based on the peak areas obtained by chromatographic analysis, and the concentration of quizalofop-p-ethyl in the samples was calculated, and then the degradation rate of each strain to quizalofop-p-ethyl within 7 d was calculated.

[0025] 1.4.3 Species identification and morphological identification of strains A drop of sterile water was dripped in the center of the glass slide. A small amount of bacterial plaque was picked with an inoculation loop and placed in the water, mixed well, and covered with a cover glass to make a smear of the quizalofop-p-ethyl-degrading strain. After standing for 5 min, it was examined under an optical microscope at 400×.

[0026] 18S rDNA sequence analysis and construction of phylogenetic tree: The purified quizalofop-p-ethyl-degrading strains were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. After obtaining the ITS sequence, it was submitted to the GenBank database to obtain the strain accession number. Through BLAST online sequence alignment, the 10 sequences with the highest homology were downloaded, and then multiple sequence alignment was performed using CLUSTALX 2.0.10, and a phylogenetic tree was constructed using MEGA5.0 to determine the species of the screened strains.

[0027] 2. Results and analysis 2.1 Primary screening of quizalofop-p-ethyl-degrading bacteria After enrichment and domestication of soil samples, the culture solution was diluted and spread on the MM+Q medium. After one week, fungal strains with different morphologies were picked and inoculated onto the RBA medium. A total of four fungal strains with different morphologies were obtained, denoted as QF1, QF2, QF3 and QF4.

[0028] 2.2 Growth of each strain during rotary culture As Figure 1, QF1, QF2, QF3, and QF4 were respectively inoculated into the MM+Q medium at a concentration of 100 mg / L and cultured at 30 °C with a constant rotation speed of 150 r / min for 7 days. White particles of quizalofop-p-ethyl precipitated at the bottom of each bottle. The most white particles were found in the CK (only MM+Q medium, without strains). Obvious clustered white hyphae were observed at the bottom of the QF2 bottle, while only a few unclustered white hyphae were visible at the bottoms of the QF1, QF3, and QF4 bottles, and no white hyphae were seen in the CK.

[0029] 2.3 Determination of the degradation ability of the initially screened strains to quizalofop-p-ethyl After ultra-high performance liquid chromatography analysis of the standard series, the regression equation of the standard curve was y = 67262x + 17559, and the correlation coefficient R 2 = 0.9989. The residual concentration of quizalofop-p-ethyl in each treatment was calculated ( Figure 2 ). Among them, the average recovery rate of the control sample was 96.69%, and the average recovery rate of quizalofop-p-ethyl in the QF4 strain sample was 96.85%, showing no significant difference from the control sample. The degradation rates of the other strains, namely QF1, QF2, and QF3, after culturing in the quizalofop-p-ethyl basic medium with an initial concentration of 100 mg / L for 7 days were 13.24%, 16.07%, and 4.78%, respectively.

[0030] 2.4 Determination of the species of the quizalofop-p-ethyl-degrading strains 2.4.1 Morphological observation and identification As Figure 3 , when QF2 was moved to an optical microscope with a magnification of 400× for microscopic examination, it could be observed that: the hyphae and sporangia were obvious. The hyphae were non-septate hyphae with conidia, and the conidiophores were relatively short (3A), and the sporangia were flask-shaped (3B). The colony morphology of QF2 on the RBA medium was as Figure 4 shown: at the initial stage of cultivation (4A), the base was slightly green, and the colony tissue was white and cottony. At the later stage of cultivation (4B), the surface of the colony was rough, the color of the base became dark brown, and the white flocculent tissue was arranged more tightly.

[0031] 2.4.2 18S rDNA sequence identification The obtained ITS sequence was submitted to the GenBank database, and the accession number of the strain was MW925693. Multiple sequence alignment of the measured sequence and 10 downloaded similar sequences was performed using CLUSTALX 2.0.10, and the result was as Figure 5 shown: there were homologous regions in each sequence, and the similarity with the measured sequence was 100%. At the same time, a phylogenetic tree was constructed using the MEGA5.0 software as Figure 6 shown. Combining the morphological characteristics of the bacteria and the results of multiple sequence alignment, it was preliminarily determined that the QF2 strain was Aspergillus fumigatus.

[0032] 3. Discussion For the first time in this experiment, it was found that Aspergillus fumigatus has the function of degrading the residual quizalofop-p-ethyl in the soil. A total of 4 fungal strains that can grow in the quizalofop-p-ethyl basal medium were screened out in this invention. By quantitatively analyzing the residual concentration of quizalofop-p-ethyl by ultra-high performance liquid chromatography, it was found that there was no significant difference in the residual concentration between QF4 and the control sample, the degradation rates of QF1 and QF3 were relatively low, and QF2 had better degradation ability than other strains under the experimental conditions of this test. However, compared with the reported bacteria that can degrade quizalofop-p-ethyl, its degradation period is long and the degradation rate is low. To make it a strain resource for the fungal agent for degrading quizalofop-p-ethyl, further research is needed on its domestication conditions, degradation principle, pathogenicity, and the preparation process of the fungal agent, etc.

[0033] 4. Conclusion In this invention, the soil with long-term application of quizalofop-p-ethyl was enriched and domesticated, and then a fungal strain QF2 that can grow with quizalofop-p-ethyl as the sole carbon source was isolated and purified. It was identified as Aspergillus fumigatus, and the degradation rate of this strain for quizalofop-p-ethyl with an initial concentration of 100 mg / L was 16.07% within 7 days. It was confirmed that this strain has the ability to degrade quizalofop-p-ethyl, providing a strain selection for using fungal agents to degrade the residual quizalofop-p-ethyl in the soil.

[0034] The above are only the preferred embodiments of the present invention and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent variations made to the above embodiments according to the technical essence of the invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A quizalofop-p-ethyl degrading strain, characterized in that, The quizalofop-p-ethyl degrading strain is Aspergillus fumigatus.

2. Use of the quizalofop-p-ethyl degrading strain as described in claim 1, characterized in that, The Aspergillus fumigatus is used for degrading quizalofop-p-ethyl.

3. The application according to claim 2, wherein The degradation rate of the Aspergillus fumigatus to quizalofop-p-ethyl with an initial concentration of 100 mg / L within 7 days is 16.07%.

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