A kind of penicillium for degrading fluocinolone and its application

Abstract

The present invention discloses a penicillium fungus for degrading fluocinolone and its application. The penicillium fungus belongs to the genus Paecilomyces ( Purpureocillium sp. ) FL-M-2 strain, which was deposited in Guangdong Provincial Microbial Culture Collection Center on January 5, 2021, with the deposit number: GDMCC No: 61418. This Penicillium has a high degradation rate for fluridone. When the environmental concentration of fluridone is 1 mg / L and 100 mg / L, it can have a good degradation effect on fluridone, and the 14d degradation rates are 50.82% and 47.86%, respectively; after the strain is applied in the field, the degradation rate of fluridone in the soil is 85.60%. This strain and its fermentation products can be widely used in the treatment of fluridone-contaminated soil and water bodies, which not only solves the problem of difficult degradation of fluridone residues in the soil after use, but also the strain and its fermentation products are safe for seed germination and seedling growth of various subsequent crops.

Description

Technical Field

[0001] The invention relates to the technical field of pesticide biodegradation, and in particular to a fluocinolone-degrading Paecilomyces pyrenoidosa and application thereof. Background Art

[0002] As the most widely used pesticide, herbicides can significantly improve agricultural productivity and crop yields. The large-scale use of herbicides may affect plant enzymes or interrupt biological systems, thereby harming or destroying normal plant growth and development, and ultimately leading to plant death; at the same time, they will inevitably enter surface water through leaching and surface runoff, and remain in the soil and crops, causing serious damage to aquatic organisms and invertebrates as well as the soil environment. In addition, compared with pesticides such as insecticides and fungicides, herbicides generally have problems such as slow degradation and long effective period. Therefore, a large amount of herbicide residues in the soil will greatly hinder the sustainable development of agricultural ecology.

[0003] Flurochloridone (FLC) is a new type of pyrrolidone soil treatment selective herbicide that can effectively control most broadleaf weeds in winter wheat, cotton, sunflower, carrot and potato fields. It has been widely promoted in recent years and was listed as a pesticide advantage development project in China in 2014. However, the applicant's research team found that: (1) the degradation dynamics and residue analysis of 25% flurochloridone emulsifiable concentrate after use in three different regions of the Qinghai Plateau (Haidong, Haixi and Haibei) showed that the half-life of flurochloridone in the soil in the three regions was 25.7 days, 27.7 days and 19.3 days, respectively. In some areas, flurochloridone (concentration of 0.013 mg / kg) was still detected in the soil 140 days after application, exceeding the maximum residue limit standard of the pesticide. Due to the unreasonable use of the pesticide, the residue detection results are even more serious. (2) Fluorochlor has a certain systemic effect. After being applied to the soil, it can be transmitted to the whole plant through the roots. The half-lives in the leaves are 8.1 days, 10.2 days and 7.0 days respectively. (3) For crops such as potatoes whose edible parts are directly exposed to the herbicide, due to the difficult degradation of fluorochlor, it not only harms human health, but also has a great impact on the planting of subsequent crops and the structure and function of soil microbial communities.

[0004] In summary: the widespread use of fluazifop in agricultural production will leave a large amount of residues in the ecological environment, which will have a certain negative impact on the development of sustainable agriculture.

[0005] Bioremediation is a technology that uses living green plants or microorganisms to remove pollutants from soil, surface water and groundwater. The residual degradation technology of herbicides based on the theory of microbial remediation is an effective way to solve the problem of soil remediation. Microbial remediation is widely used in the degradation research of the herbicide atrazine residue in agricultural production. Different types of microorganisms (bacteria, fungi) with atrazine biodegradability have been identified and isolated in large quantities and widely used in the removal of atrazine residues. Mixed microorganisms composed of iron-oxidizing bacteria, coriolus versicolor and white rot fungi can also effectively degrade atrazine, and the degradation rate can even reach 98%. In recent years, there have been many studies on the microbial degradation of herbicides, among which the research on the degradation bacteria of sulfonylurea, chloroacetamide, imidazolinone and triazine herbicides has made breakthrough progress, but the research on the biodegradation products and technologies of the pyrrolidone herbicide flurazepam is almost blank.

[0006] In summary, as a herbicide, fluazifop has a very good control effect on various weeds in the field, but it degrades slowly in the environment, and has a great impact on the residual pesticide damage to subsequent crops. It is urgent to develop a technical method that can quickly degrade fluazifop, and utilizing the degradation metabolism of microorganisms in the natural environment is an efficient and ideal way to eliminate and repair herbicide residues. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a fluocinolone-degrading bacterial agent and application thereof.

[0008] The technical solution adopted by the present invention is:

[0009] The Paecilomyces spp. used to degrade fluocinolone of the present invention is a species of Paecilomyces spp. Purpureocillium sp. ) FL-M-2 strain, whose 16S rDNA and 28S rDNA gene intergenic sequence (ITS sequence) is shown in SEQ ID NO: 1, was deposited in Guangdong Provincial Microbiological Culture Collection Center on January 5, 2021, with the deposit number: GDMCC No: 61418, and the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0010] The Paecilomyces oxytoca can be used to degrade the herbicide fluridone. Specifically, it can be used to prepare a preparation for degrading the pesticide residue of the herbicide fluridone in soil or water.

[0011] The present invention also provides a fluocinolone-degrading bacterial agent, which comprises the Paecilomyces pyogenes of the present invention or its fermentation liquid.

[0012] Beneficial effects of the present invention:

[0013] The Penicillium spp. provided by the invention has a high degradation rate for fluocorochlor, and has a good degradation effect on fluocorochlor when the environmental concentration of fluocorochlor is 1 mg / L (low concentration) and 100 mg / L (high concentration), and the 14-day degradation rates are 50.82% and 47.86% respectively. The strain and its fermentation product can be widely used in the treatment of fluocorochlor contaminated soil and water bodies, and not only solves the problem that the residue of fluocorochlor in the soil after use is difficult to degrade, but also the strain and its fermentation product are safe for seed germination and seedling growth of various subsequent crops.

[0014] In addition to the above-described objects, features and advantages, the present invention has other objects, features and advantages. The present invention will be further described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The drawings constituting a part of this application are used to provide a further understanding of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0016] Figure 1 The isolated and trained strain of the embodiment of the present invention;

[0017] Figure 2 is a microscope image (40 times) of the FL-M-2 strain of the present invention;

[0018] Figure 3 Is a phylogenetic tree of the FL-M-2 strain of the present invention embodiment;

[0019] Figure 4 is the strain growth curve of the embodiment of the present invention;

[0020] Figure 5 This is the degradation rate curve of low concentration (1 mg / L) fluazifop by the FL-M-2 strain of the embodiment of the present invention;

[0021] Figure 6 This is the degradation rate curve of the FL-M-2 strain of the embodiment of the present invention to low concentration (100 mg / L) fluazifop;

[0022] Figure 7 This is the safety evaluation of the fermentation liquid of the FL-M-2 strain of the present invention on spring wheat seed germination and seedling growth;

[0023] Figure 8 This is a safety evaluation of the FL-M-2 strain fermentation liquid of the embodiment of the present invention on spring rapeseed seed germination and seedling growth. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. 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.

[0025] Example 1

[0026] Strain isolation, identification and growth curve determination

[0027] Sample collection: The surface soil of potatoes where fluridone was used in large quantities for a long time was collected from the experimental field of the Institute of Plant Protection, Qinghai Academy of Agriculture and Forestry Sciences, and stored at -20℃ for future use.

[0028] Isolation and purification of bacteria:

[0029] S1: Weigh 10 g of soil sample and place it in a 250 mL Erlenmeyer flask containing 90 mL of sterile water. Oscillate at 28°C and 180 r / min for 30 min to prepare a bacterial suspension diluted 10 times. Pipette 1 mL of the bacterial suspension into a test tube containing 9 mL of sterile water to dilute it to 10. -2 , and then serially dilute 10 times in this way until 10 -6 .

[0030] S2: Spread the diluted bacterial suspension on modified Martin medium and culture at 28℃ for 5-7d. Pick a single colony from the plate and repeat the streak isolation culture several times until a single colony is isolated. Store the single colony in the corresponding solid slant modified Martin medium; wherein, the modified Martin medium: 2 g yeast extract powder, 20 g glucose, 5 g peptone, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, pH 6.4±0.2, sterilize at 121℃ for 30 min; add 1.5% agar to the solid medium.

[0031] Acclimation: prepare 500 mL of inorganic salt solid culture medium, sterilize under high pressure, and when the temperature is cooled to about 40°C, absorb 5 mL of 10 mg / L fluridone standard mother solution to prepare an inorganic salt culture medium with a fluridone concentration of 0.1 mg / L. Inoculate the isolated and purified strain on this plate and culture it at 28°C for 2-3 days. Similarly, transfer the strain grown in the fluridone inorganic salt culture medium with a concentration of 1.0 mg / L to the inorganic salt solid culture medium with a fluridone concentration of 10.0 mg / L, continue acclimation and transfer culture, and continuously increase the concentration. When the fluridone concentration increases to 100.0 mg / L, if it can still grow on the plate, it means that this strain has the potential to degrade fluridone, and the strain is preserved for identification.

[0032] The strains that survived the training were graded diluted and spread on the plates, and the strains with different colony appearance characteristics were selected for purification. The strains were obtained in the fluocinolone inorganic salt medium and were able to grow and grow well. Figure 1 shown.

[0033] Microscope observation: The cell morphology of the domesticated strain with the potential to degrade fluazifop was observed under a microscope, such as Figure 2 shown.

[0034] Nucleotide sequence determination: The recombinant plasmid was sequenced by Sangon Biotechnology (Shanghai) Technology Service Co., Ltd. After obtaining the sequence number, log in to the NCBI website, enter the Blast interface, select the homologous sequence closest to the known strain sequence, and download it for backup. Then use MEGA7.0 software to construct the phylogenetic tree, such as Figure 3 As shown, based on the phylogenetic tree analysis results combined with the strain morphological characteristics, strain FL-M-2 was determined to be Purpureocillium sp.

[0035] Strain growth curve determination: The screened degradation strains were cultured at 28°C and 180 r / min. Samples were taken every 1 day, and 100 μL of bacterial solution was sampled each time. The OD value of the bacterial solution was measured using an enzyme marker. 620 The absorbance value under the condition of 100 μm was used to draw the growth curve of the strain (see Figure 4 ).

[0036] Example 2

[0037] Test of the degradation ability of strains under low concentration of fluazifop

[0038] Chromatographic detection conditions:

[0039] Chromatographic column, C18 column; column temperature, 40°C; mobile phase A was methanol, mobile phase B was 0.1% acetic acid + 5mmol / L ammonium acetate aqueous solution; flow rate was 0.25mL / min; injection volume, 1.0µL.

[0040] Determination of additive recovery rate:

[0041] Fluorochloridone standard was added to three culture media respectively, with concentrations of 0.01 mg / L, 0.1 mg / L, and 1 mg / L, and five replicates were performed. 10 mL of modified Martin liquid medium was taken and placed in a 50 mL stoppered centrifuge tube. 10 mL of acetonitrile solution was added and vortexed for 3 min to mix thoroughly. 3 g of NaCl was added, vortexed for 1 min, centrifuged at 5000 r / min for 5 min, 1.5 mL of supernatant was taken, 50 mg of PSA and 100 mg of anhydrous magnesium sulfate were added, vortexed for 1 min, centrifuged at 12000 r / min for 2 min, and the supernatant was taken. After filtering through a 0.22 µm membrane, it was determined by ultra-high performance liquid chromatography-tandem mass spectrometry.

[0042] Add recovery rate calculation formula:

[0043] Addition recovery rate (%) = spiked amount / spike sample measured value × 100

[0044] The results showed that the peak area linear relationship of the standard sample was good within the concentration range, and the average recovery rate of fluridone in the modified Martin liquid medium was in the range of 83.1%-97.6%, indicating that the method for determining the content of fluridone in the modified Martin liquid medium was reliable, simple and reliable to operate, and met the technical requirements for quality control of pesticide residue analysis.

[0045] Determination of the degradation ability of strains

[0046] The strains with the potential to degrade flurochloridone obtained by domestication were inoculated into the modified Martin liquid medium and cultured at 28°C and 180r / min for 24h as seed solution. The seed solution was inoculated into the 1mg / L flurochloridone inorganic salt liquid medium at a 2% inoculation amount (ratio of seed solution volume to culture solution volume), and cultured at 28°C and 180r / min. Samples were taken every 1d, 3d, 5d, 7d, and 14d, and the inorganic salt medium with the same concentration of flurochloridone but not inoculated was used as the control. The degradation rate of flurochloridone by each strain was detected and analyzed by liquid chromatography-tandem mass spectrometry. 10mL of culture solution was taken and placed in a 50mL stoppered centrifuge tube, and 10mL of acetonitrile solution was added. Vortex and oscillate for 3min to mix thoroughly. Add 3g NaCl, vortex for 1min, centrifuge at 5000r / min for 5min, take 1.5mL of supernatant, add 50mg PSA and 100mg anhydrous magnesium sulfate, vortex for 1min, centrifuge at 12000r / min for 2min, take the supernatant, filter through 0.22µm membrane, and then provide it for ultra-performance liquid chromatography-tandem mass spectrometry.

[0047] Degradation rate calculation formula:

[0048] X%=(A1-A2) / A1×100;

[0049] Where:

[0050] X%—degradation rate of fluazifop;

[0051] A1—pesticide concentration in control (mg / L);

[0052] A2—pesticide concentration in treatment (mg / L);

[0053] The results showed that the degradation rate of fluazifop-butyl to the strain of the present invention at an environmental concentration of 1 mg / L was Figure 5 The degradation rates of strain FL-M-2 at 1d, 3d, 5d, 7d and 14d were 11.28%, 25.67%, 31.83%, 39.63% and 50.82% respectively; while the natural degradation rates of the blank control without adding bacterial agent were 2.58%, 2.69%, 4.39%, 4.50% and 5.76%.

[0054] Example 3

[0055] Test of the degradation ability of strains under high concentration of fluazifop

[0056] The strains with the potential to degrade flurochloridone obtained by domestication were inoculated into the modified Martin liquid medium and cultured at 28°C and 180r / min for 24h as seed solution. The seed solution was inoculated into the 100mg / L flurochloridone inorganic salt liquid medium at a concentration of 2% (ratio of seed solution volume to culture solution volume), and cultured at 28°C and 180r / min. Samples were taken every 1d, 3d, 5d, 7d, and 14d, and the inorganic salt medium with the same concentration of flurochloridone but not inoculated was used as the control. The degradation rate of flurochloridone by each strain was detected and analyzed by liquid chromatography-tandem mass spectrometry. 10mL of culture solution was taken and placed in a 50mL stoppered centrifuge tube, and 10mL of acetonitrile solution was added. Vortex and oscillate for 3min to mix thoroughly. Add 3g NaCl, vortex for 1min, centrifuge at 5000r / min for 5min, take 1.5mL of supernatant, add 50mg PSA and 100mg anhydrous magnesium sulfate, vortex for 1min, centrifuge at 12000r / min for 2min, take the supernatant, filter through 0.22µm membrane, and then provide it for ultra-performance liquid chromatography-tandem mass spectrometry.

[0057] Degradation rate calculation formula:

[0058] X%=(A1-A2) / A1×100;

[0059] Where:

[0060] X%—degradation rate of fluazifop

[0061] A1—Pesticide concentration in control (mg / L)

[0062] A2—Pesticide concentration in treatment (mg / L)

[0063] The results show that the degradation rate of the strain of the present invention for fluazifop in an environmental concentration of 100 mg / L is Figure 6 The degradation rates of strain FL-M-2 at 1d, 3d, 5d, 7d and 14d were 10.46%, 24.35%, 29.68%, 34.46% and 47.86%, respectively, while the natural degradation rates of the blank control without adding bacterial agent were 1.77%, 2.28%, 2.79%, 4.03% and 4.40%.

[0064] Example 4

[0065] Safety of degradation strains and their fermentation liquid to crops

[0066] The seed germination method was used. Under the culture conditions of 28℃ and 180 r / min, a single colony of strain FL-M-2 was picked from the plate and inoculated into the modified Martin liquid medium for 5 days. A certain amount of the fermentation liquid of the strain was taken, and distilled water was used as the solvent to prepare the fermentation liquid, 10-fold diluted fermentation liquid and 30-fold diluted fermentation liquid. Wheat and rapeseed seeds were used as the test objects. 16 seeds were sown in each culture dish, and 5 mL was added to each dish with a pipette. Five treatments were set up, namely, water treatment (CK), medium without bacteria treatment, CK (M), fermentation liquid, 10-fold diluted fermentation liquid, and 30-fold diluted fermentation liquid. Each treatment was repeated 3 times. The culture was carried out in an intelligent light incubator at 25℃ constant temperature and moisturizing light, with 12 h of light and 12 h of darkness. The seeds were sterilized before the bioassay, and then distilled water was added to germinate until they turned white. After 7 days, the root length, shoot length, fresh weight and dry weight of each wheat and rapeseed seed were calculated.

[0067] The results showed that the root length, shoot length and dry weight of wheat and rapeseed seeds treated with 30-fold diluted fermentation solution of strain FL-M-2 had no significant difference with those treated with CK. The root length, shoot length, fresh weight and dry weight of wheat and rapeseed seeds treated with the original solution of the strain were lower than those of other treatments, and the difference was significant at the 0.05 level. There was no significant difference in the root length, shoot length, fresh weight and dry weight between the fermentation solution treatment and the culture medium without bacteria treatment CK(M), indicating that the fermentation solution of strain FL-M-2 diluted 30 times had no effect on the growth of seed seedlings, and the original solution of the strain had no promoting effect on the growth of seed seedlings (see Figure 7 and Figure 8 ).

[0068] Example 5

[0069] Field test of degradation efficiency of degradation strains

[0070] The field plot method was used to conduct the experiment in potato fields. Under the same experimental conditions, after sowing potatoes, the effective ingredient 750a.ig / hm 2 Spraying fluchlorhydrone, the plot area is 20m 2 , repeated 4 times, a total of 8 plots, randomly arranged; the screened degradation strains were cultured in improved Martin liquid medium at 28°C and 180r / min for 7 days, and then 600mL was sprayed on 4 treatment plots in each plot. The other 4 plots were used as a control with clear water. Soil samples (0-10cm) were collected every 7 days, mixed evenly and brought back to the laboratory for testing.

[0071] The results are shown in Table 1. The original deposition of flurochloridone in the degradation bacteria treatment area was 0.9902 mg / L, and the original deposition of flurochloridone in the clear water control area was 1.0832 mg / L. After 28 days, the residual amount of flurochloridone in the degradation bacteria treatment area was 0.1426 mg / L, and the degradation rate was 85.60%; while the residual amount of flurochloridone in the clear water blank control area was 0.5931 mg / L, and the natural degradation rate was 45.25%. The results show that the degradation strain FL-M-2 accelerated the degradation of flurochloridone under natural field conditions. Since it was collected from the local habitat field and applied to the field soil through biostimulation, it has good field colonization ability and can accelerate the degradation rate of flurochloridone.

[0072] Table 1 Residues of fluazifop in soil treated with degradation strains and blank control

[0073]

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Sequence Listing <110> Qinghai Academy of Agriculture and Forestry Sciences <120> A kind of penicillium for degrading fluocinolone and its application <130> 1 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 573 <212> DNA <213> Purpureocillium sp. <400> 1 ttgatatgct taagttcagc gggtattcct acctgatccg aggtcaactc taagaaagtt 60 gggcgtttta cggcgtgacc gcctccgcgc tccggtgcga ggtgtgtgct actacgcagg 120 ggaggctgcg gcggggtcgc cactgcattt cggggcggc tggtgtgccg tcccccaaca 180 ccgaggcccc cggggggggc tcgagggttg aaatgacgct cgaacaggca tgcccgccag 240 aatgctggcg ggcgcaatgt gcgttcaaag attcgatgat tcactgaatt ctgcaattca 300 cattacttat cgcatttcgc tgcgttcttc atcgatgcca gaaccaagag atccgttgtt 360 gaaagttttg attcatttgt ttttgcttgt gcaactcaga gaagaaattc cgcccgctgg 420 gcgtaatgca agagagtttg gggtccctgc ggcgggcgcc tgggtccggc gccggcgcgg 480 gggcaggcgg ccggggcgtt cccgccgagg caactgaggt aaggttcaca gtgggtttgg 540 gagttgtata actcggtaat gatccctccg foot 573

Claims

1. A Paecilomyces spp. for degrading fluocinolone, characterized in that: The Paecilomyces spp. is a species of the genus Paecilomyces ( Purpureocillium sp. ) FL-M-2 strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on January 5, 2021, with the deposit number: GDMCC No: 61418.

2. The use of Paecilomyces as claimed in claim 1, characterized in that: The Paecilomyces spp. is used to degrade the herbicide fluridone.

3. The use according to claim 2, characterized in that: The Penicillium is used for preparing a preparation for degrading the pesticide residue of fluocinolone in soil or water.

4. A fluocinolone-degrading bacterial agent, characterized in that: The invention comprises the Paecilomyces spp. or its fermentation liquid as claimed in claim 1.

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