Method for degrading triadimenol in soil by virtue of novel microbial strain

[0036] Embodiment 1: the screening of new bacterial strain E-9

[0037] The present invention collects the activated sludge of the aeration tank from a pesticide factory in Zhejiang Province, and rapidly screens bacterial species from it within 24 hours; the specific method of screening is: take 10g of activated sludge, place it in 100mL enrichment medium, and simultaneously Add 0.1mL of 100g/L triadimenol original drug to make the final concentration 100mg/L, and at 30℃, 180r.min -1 Culture on a shaking table, inoculate once every 7 days thereafter, inoculate into fresh medium with 10% inoculum, and gradually increase the concentration of the tested pesticide to 600 mg/L with 100 mg/L as an increase unit. Then the culture solution was added to the inorganic salt medium with the tested pesticide triadimenol (600mg/L) as the only carbon source for acclimatization 2-5 times, 7 days each time, and finally separated on the ordinary medium by smearing. Select colonies with fast growth, regular colonies, and stable passage, and purify them on ordinary medium plates for 2 or 3 times. After microscopic examination, they show a single form to obtain the strains for primary screening. The first screened strains were tested for degradation performance in the degradation medium, and according to the degradation rate of triadimenol, the strain E-9 with significant degradation ability was screened out as the starting strain for further research;

[0038] The formulation of the enriched medium is: adding triadimenol solutions of different mass concentrations to the ordinary medium, so that the final mass concentrations of triadimenol are 100, 200, 300, 400, 500, and 600 mg/L respectively;

[0039] Described domestication inorganic salt culture medium is: (NH 4 ) 2 SO 4 1.0g, FeSO 4 ·7H 2 O 0.005g, CaSO 4 0.08g, Na 2 MoO 4 2H 2 O 0.0033g, MgSO 4 0.2g, K 2 HPO 4 1.0g, KH 2 PO 4 1.0g, distilled water 1000mL, pH value 7, triadimenol 600mg/L;

[0040] The fermentation medium formula is: (NH 4 ) 2 SO 4 1.0g, FeSO 4 ·7H 2 O 0.005g, CaSO 4 0.08g, Na 2 MoO 4 2H 2 O 0.0033g, MgSO 4 0.2g, K 2 HPO 4 1.0g, KH 2 PO 4 1.0g, 3.0g sucrose, 0.5g peptone, 1000mL distilled water, pH 7.0.

[0041] Embodiment 2: Identification of new bacterial strain E-9

[0042] For the strain E-9 screened in Example 1, the strain collection center of Wuhan University was entrusted with the measurement and analysis of the 16SrRNA gene sequence of the microbial strain, the observation of the morphology of the strain, and the identification of the physiological and biochemical characteristics of the microbial strain. According to the above test results, strain E-9 was identified as Achromobacterxylosoxidans, Gram-negative, and this strain was preserved in Wuhan China Type Culture Collection Center on June 24, 2016. The number is CCTCCNO:M2016348.

[0043] Strain E-9CCTCC NO:M 2016348 16S rRNA gene sequence:

[0044] GGCACGATCTACGTGGTATCGCCCCCCTTGCGGTTAGGCTAACTACTTCTGGTAAAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGCGACATGCTGATCCGCGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATCCGGACTACGATCGGGTTTCTGGGATTGGCTCCCCCTCGCGGGTTGGCGACCCTCTGTCCCGACCATTGTATGACGTGTGAAGCCCTACCCATAAGGGCCATGAGGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCTTTCGTAGCAACTAATGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTTCCGGTTCTCTTGCGAGCACTGCCAAATCTCTTCGGCATTCCAGACATGTCAAGGGTAGGTAAGGTTTTTCGCGTTGCATCGAATTAATCCACATCATCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGTCAACTTCACGCGTTAGCTGCGCTACCAAGGCCCGAAGGCCCCAACAGCTAGTTGACATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTGCATGAGCGTCAGTGTTATCCCAGGAGGCTGCCTTCGCCATCGGTGTTCCTCCGCATATCTACGCATTTCACTGCTACACGCGGAATTCCACCTCCCTCTGACACACTCTAGCCCGGTAGTTAAAAATGCAGTTCCAAAGTTAAGCTCTGGGATTTCACATCTTTCTTTCCGAACCGCCTGCGCACGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTATTCTGCAGGTACCGTCAGTTT CGCGGGGTATTAACCCACGACGTTTCTTTCCTGCCAAAAGTGCTTTACAACCCGAAGGCCTTCATCGCACACGCGGGATGGCTGGATCAGGGTTTCCCCCATTGTCCAAAATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCTGGTCGTCCTCTCAAACCAGCTACGGATCGTCGCCTTGGTGAGCCGTTACCCCACCAACTAGCTAATCCGATATCGGCCGCTCCAATAGTGCAAGGTCTTGCGATCCCCTGCTTTCCCCCGTAGGGCGTATGCGGTATTAGCTACGCTTTCGCGTAGTTATCCCCCGCTACTGGGCACGTTCCGATACATTACTCACCCGTTCGCCACTCGCCACCAGACCGAAGTCCGTGCTGCCGTTCGACTGCATG

[0045] Table 1 Physiological and biochemical characteristics of strain E-9 - enzyme activity, carbon source assimilation

[0046]

[0047]

[0048] +: Positive reaction; -: Negative reaction

[0049] Table 2 Physiological and biochemical characteristics of strain E-9—acid production using carbon source

[0050]

[0051] +: Positive reaction; -: Negative reaction

[0052] Embodiment 3: Utilize strain E-9 to degrade the method 1 of triadimenol in soil

[0053] Follow these steps:

[0054] (1) Strain activation medium: yeast powder 5.0g, peptone 10.0g, NaCl 5.0g, distilled water 1000mL, pH7.0. After being sterilized, it is ready for use;

[0055] (2) Strain fermentation medium: (NH 4 ) 2 SO 4 1.0g, FeSO 4 ·7H 2 O 0.005g, CaSO 4 0.08g, Na 2 MoO 4 2H 2 O 0.0033g, MgSO 4 0.2g, K 2 HPO 4 1.0g, KH 2 PO 4 1.0g, 3.0g sucrose, 0.5g peptone, 1000mL distilled water, pH 7.0. After being sterilized, it is ready for use;

[0056] (3) Preparation of seed liquid: Pick the preserved strain E-9CCTCC NO:M 2016348 of 1-ring Achromobacterxylosoxidans slant, insert it into the medium of step (1), and put it under the conditions of 30°C and 180rpm After 24 hours of shaking culture, the seed liquid was obtained and set aside;

[0057] (4) Preparation of the degrading bacterial agent: insert the seed solution of step (3) into the medium of step (2) according to the inoculation amount of 5% by volume; obtain the degrading bacterial agent after shaking and culturing at 30° C. and 180 rpm for 48 hours, and set aside;

[0058] (5) Add the degrading bacteria agent in step (4) into the soil samples containing 5mg/kg, 25mg/kg, and 50mg/kg triadimenol respectively in a ratio of 10%, add sterile water, and keep the soil humidity at the field water holding capacity About 60% of the concentration, mix well to make the triadimenol and bacterial agent evenly distributed. Add sterile water on time, try to keep the soil moisture at about 60% of the field water holding capacity, and take the soil sample with the same mass concentration of triadimenol without bacteria agent as the control. Degradate at 30°C in the dark for 7 days; measure the residual amount of triadimenol in the soil at 7 days.

[0059](6) Determination of triadimenol residues in soil samples: adopt high performance liquid chromatography to analyze the soil samples of step (5) triadimenol residues, soil samples with 100mL acetone/distilled water (V/V, 5/ 1) After shaking and extracting for 2 hours, filter, and wash the filter residue with 40 mL of acetone/distilled water (V/V=5/1) for 3 times, and combine the filtrates on a rotary evaporator to remove the acetone under reduced pressure. Add 50 mL of saturated aqueous sodium chloride solution to the concentrated solution, extract with 50, 40 and 30 mL of dichloromethane in sequence, combine the extracts, and concentrate to about 2 mL. Add 2g of anhydrous sodium sulfate, 6g of florisil and 2g of anhydrous sodium sulfate to the glass chromatography column successively, transfer the extract to the column, and use 50mL of dichloromethane/acetone (V/V=9/1) Rinse, collect the eluate, concentrate to 1mL, set the volume to 10mL with methanol, and carry out high-performance liquid chromatography detection conditions: detection wavelength 223nm, mobile phase is methanol: water (V:V)=70:30, and the flow rate is 0.8mL/min, using a SUPELCO C18 column (250mm×4.6mm, 5μm), the injection volume is 20μL, and the column temperature is 25°C. The peak eluting time of triadimenol was 7.12min. The content of triadimenol in different triadimenol-added soil samples (5mg/kg, 25mg/kg, 50mg/kg) was determined to be 4.79mg/kg, 24.31mg/kg, 48.12mg/kg respectively on day 0, and the control soil at 7 days The triadimenol residues in the samples were 4.63mg/kg, 23.40mg/kg, 47.60mg/kg respectively, and the triadimenol residues in the inoculant soil samples were 3.40mg/kg, 17.89mg/kg, 36.56mg/kg respectively. kg, calculated according to the formula of strain degradation rate (%)=(uninoculated soil triadimenol content-inoculated soil triadimenol content)/uninoculated soil triadimenol content*100, triadimenol degraded in control soil The rates were 3.34%, 3.74%, and 1.08%, respectively, and the degradation rates of the bacteria agents to triadimenol were 29.03%, 26.41%, and 24.02%, respectively, see Figure 4.