CN112481170B - Pymetrozine degrading bacterium IURM F18 and application thereof - Google Patents
Pymetrozine degrading bacterium IURM F18 and application thereof Download PDFInfo
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Abstract
The invention discloses a pymetrozine degrading bacterium IURM F18 and application thereof, belonging to the technical field of biology. Experiments prove that the pymetrozine degrading bacterium IURM F18 can survive in an environment with pymetrozine as a unique carbon source and has high pymetrozine degrading activity. The pymetrozine degrading bacterium IURM F18 provided by the invention can be applied to the degradation of pymetrozine pesticide in the polluted environment, and has good application value in the aspects of soil pollution treatment and environment restoration.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to pymetrozine degrading bacteria IURM F18 and application thereof.
Background
Chemical pesticides play an important role in modern agricultural production as an important means for reducing loss and ensuring agricultural harvest. However, due to the long-term unreasonable use of chemical pesticides, the storage amount of the chemical pesticides in the environment is higher and higher, and serious pollution is caused to the ecological environment. Wherein, pesticide residue in agricultural products seriously exceeds standard and forms direct threat to human body safety and health.
At present, pesticide degradation methods are numerous at home and abroad, and biodegradation is one of the most important methods among different degradation methods. The microbial repairing method is to utilize the metabolism of specific microbe to absorb, convert, eliminate or degrade the effective components of pesticide to degrade pesticide and purify environment. At present, the technology is considered by experts of relevant industry departments at home and abroad to be the most valuable and vital treatment technology in the field of ecological environment protection, is a main treatment means for remedying agricultural pollution in the future and is also a treatment means for effectively solving the ecological remediation of chemical pesticide polluted soil.
Pymetrozine belongs to pyridine (pyridine azomethine) or triazone insecticides, is a brand-new non-biocidal insecticide, has contact poisoning effect on pests, and has systemic activity. The insecticidal composition is widely applied to preventing and treating various pests of aphididae, plant hopper, aleyrodidae, leafhopper and the like on vegetables, wheat, rice, cotton and fruit trees in agricultural planting production in China. The mechanism of action is that aphids or plant hoppers almost immediately produce a needle-blocking effect upon contact with pymetrozine, immediately stop feeding and finally starve to death, and the process is irreversible. Pymetrozine can be transported in the xylem and phloem of the plant body; therefore, the fertilizer can be used for foliar spraying and soil treatment. Due to the good transmission and conduction characteristics, branches and leaves newly grown after stem and leaf spraying can be effectively protected. Pymetrozine is favored because of its excellent effect of controlling pests in the field, strong selectivity and no direct poisoning property, but recently, the Environmental Protection Agency (EPA) has classified pymetrozine as a "possible" carcinogen for human beings, and therefore, it is urgent to find a method for degrading pymetrozine pesticide economically and hygienically.
Disclosure of Invention
The invention aims to provide pymetrozine degrading bacteria IURM F18 and application thereof in the aspects of pymetrozine degradation in soil and water and the like.
In order to solve the technical problems, the invention provides the following technical scheme: a pymetrozine degrading bacterium IURM F18; wherein the pymetrozine degrading bacterium IURM F18 is numbered as follows: IURM F18 has been deposited in China general microbiological culture Collection center (CGMCC) at 12.10.2020, the address of the collection unit is No. 3 of West Lu No. 1 of Beijing, the south-facing area of the republic of Yangxi, the collection number is CGMCC No.20853, and the collection is classified and named as sphingosine halophilus (Sphingobacterium thallophilum).
The pymetrozine-degrading bacterium IURM F18 has a repairing effect on an environment polluted by pymetrozine, and particularly has a repairing effect on a soil environment polluted by pymetrozine and a water body polluted by pymetrozine.
The pymetrozine degrading bacteria IURM F18 have a degrading effect on waste pymetrozine in soil and pymetrozine contained in a water body, and can be directly applied to the field of purification treatment of farmlands.
The screening method of the pymetrozine-degrading bacterium IURM F18 comprises the following steps:
1) Weighing soil from sludge polluted by pymetrozine, adding pymetrozine-degrading bacteria IURM F18 to screen liquid culture medium for enrichment, and culturing in a constant temperature shaking table at 35-40 deg.C and 150-200r/min for 4-10 days;
2) Absorbing the culture medium obtained in the step 1, adding a new pymetrozine degrading bacterium IURM F18 screening liquid culture medium, and carrying out passage for 3-6 times;
3) Streaking and separating the bacterial liquid obtained in the step (2) on an LB (lysostaphin-based) flat plate to obtain pymetrozine degrading bacteria IURM F18;
wherein the pymetrozine degrading bacteriaThe IURM F18 screening medium comprises the following components: every 1L of deionized water contains 100mg/L of pymetrozine pesticide, 0.66g of ammonium chloride and dipotassium hydrogen phosphate (K) 2 HPO 4 ) 1.5g of potassium dihydrogen phosphate (KH) 2 PO 4 ) 0.5g magnesium sulfate (MgSO) 4 ) 0.1g of dipotassium hydrogen phosphate (K) 2 HPO 4 ) 1.5g, 0.35g of sodium chloride, and pH 7.0-7.2;
the LB solid medium comprises the following components: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 15g of agar powder, and the volume is made up to 1L by deionized water.
The preferable screening method of the pymetrozine-degrading bacterium IURM F18 comprises the following steps:
1) Weighing 5g of soil from sludge polluted by pymetrozine, adding 100mL of pymetrozine degrading bacteria IURM F18 screening liquid culture medium for enrichment, and culturing in a constant temperature shaking table at 35 ℃ and 180r/min for 5-7 days;
2) 3mL of the culture medium obtained in the step 1 is sucked and added with a new pymetrozine degrading bacterium IURM F18 screening liquid culture medium for passage, and the passage is carried out for 4-5 times;
3) Streaking and separating the bacterial liquid obtained in the step (2) on an LB (lysostaphin-based) flat plate to obtain pymetrozine degrading bacteria IURM F18;
the dry powder of the pymetrozine-degrading bacteria IURM F18 is prepared by drying the pymetrozine-degrading bacteria IURM F18 subjected to amplification culture by a conventional method.
The pymetrozine degrading bacterium IURM F18 is obtained by screening according to the following method:
1. weighing an earth separation strain from sludge polluted by pymetrozine in Changzhou Wuji area of Jiangsu, weighing 5g of sludge, adding 100mL of pymetrozine degrading bacteria IURM F18 screening liquid culture medium for enrichment, and culturing for 5-7 days in a constant temperature shaking table at 35 ℃ and 180 r/min; then 3mL of culture medium is sucked and added with a new pymetrozine degrading bacterium IURM F18 screening liquid culture medium for passage, the passage is carried out for 4-5 times, streaking separation is carried out on an LB (Langmuir-Blodgett) plate, and a batch of strains which can survive in the culture medium taking pymetrozine as a unique carbon source are obtained finally after hundreds of separation test screening;
the pymetrozine degrading bacteria IURM F18 screening culture medium comprises the following components: 1L of deionized water contains pymetrozine pesticide100mg/L, ammonium chloride 0.66g, dipotassium hydrogen phosphate (K) 2 HPO 4 ) 1.5g of potassium dihydrogen phosphate (KH) 2 PO 4 ) 0.5g magnesium sulfate (MgSO) 4 ) 0.1g of dipotassium hydrogen phosphate (K) 2 HPO 4 ) 1.5g, 0.35g of sodium chloride, and pH 7.0-7.2;
the LB solid medium comprises the following components: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 15g of agar powder, and the volume is made up to 1L by deionized water.
2. Analyzing the degradation efficiency of the different strains obtained by separation on the pymetrozine in the culture medium, and screening to obtain the strain with the best pymetrozine degradation effect.
The specific determination method is as follows:
1. preparation of the Medium
Basic salt culture medium: NH (NH) 4 Cl 0.66g,K 2 HPO 4 1.5 g,KH 2 PO 4 0.5 g,MgSO 4 0.1 g,K 2 HPO 4 1.5g, naCl 0.35g, pH 7.0-7.2; sterilizing at 121 deg.C for 20min.
LB liquid medium: 5g of yeast powder, 10g of sodium chloride, 10g of peptone and 1000mL of deionized water, wherein the components are sterilized at 121 ℃ for 20min.
2. Method for measuring pymetrozine degradation efficiency
2.1 preparation of Standard Curve of pymetrozine
Dissolving 0.0256g of pymetrozine standard substance with chromatographic acetonitrile, and making the volume constant to a 50mL volumetric flask to obtain 500mg/L mother liquor. Diluting the mother liquor with chromatographic pure acetonitrile, preparing series of standard solutions with concentrations of 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1 and 0.2mg/L step by step, and measuring peak area by using a high performance liquid chromatograph under the condition of wavelength of 298nm to obtain a concentration-peak area standard curve.
2.2 method for measuring pymetrozine degradation rate
And (3) taking the culture solution, measuring the peak area by using a high performance liquid chromatograph under the condition of 298nm wavelength, calculating the pymetrozine concentration by using a linear regression equation established by a pymetrozine standard curve, and further calculating to obtain the pymetrozine degradation rate. Three replicates were set up for all experiments.
2.3 obtaining the optimal strain through the degradation rate of the pymetrozine by the pymetrozine degrading bacteria IURM F18
Culturing pymetrozine-degrading bacteria IURM F18 strain in LB liquid culture medium, centrifugally collecting bacteria, re-suspending with sterile water, and diluting to OD 600 =1, inoculating the selected strain to 1-100.0 mL of a basic liquid inorganic salt culture medium containing 100mg/L pymetrozine, placing the culture medium in a constant temperature shaking table for culture at 35 ℃ at 180r/min, and sampling every 6 hours to determine the degradation rate.
Wherein the pymetrozine degrading bacteria IURM F18 have obvious effect on the degradation of pymetrozine, the strain grows well, and the degradation rate of the pymetrozine within 30 hours is 91.33%;
2.4 degradation of waste pymetrozine in soil by pymetrozine degrading bacterium IURM F18
Inoculating 1% of pymetrozine-degrading bacteria IURM F18 into 100g of sterilized soil, measuring to obtain the content of pymetrozine in the soil of 58mg/Kg, culturing for 30h at the constant temperature of 35 ℃, and measuring the degradation rate of pymetrozine to be 77.01%.
2.5 degradation of waste pymetrozine in wastewater by pymetrozine-degrading bacterium IURM F18
Inoculating 1% of pymetrozine-degrading bacteria IURM F18 into 100mL of pymetrozine-containing wastewater, determining that the pymetrozine content of the wastewater is 58mg/L, culturing for 30h at 35 ℃ and 180r/min, and determining that the pymetrozine degradation rate is as follows: 81.23 percent.
Identification of strains
Obtaining the genome DNA of pymetrozine degrading bacteria IURM F18 by adopting an oscillation crushing method, amplifying the 16S rRNA gene of the strain by a PCR method, and sending the amplified gene to Shanghai bioengineering companies for sequencing analysis; the upstream primer sequences used to amplify the 16S rRNA gene were: 5 'AGAGAGTTTGATCCTGGCTCAG 3' and the downstream primer sequence is 5'GGTTACCTTGTTACGACTT 3'.
The PCR reaction conditions were: pre-denaturation at 98 ℃ for 5min, denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, and extension at 72 ℃ for 40s, wherein 30 cycles of reaction are set, and the temperature is kept at 10 ℃ for 5min. Using the BLAST function of NCBI for sequence alignment in the GeneBank database, the classification of the analyzed strains was: sphingosine bacilli halophilus (S.thallophilum)
The gene sequence of IURM F1816S rRNA of pymetrozine degrading bacteria is shown in a sequence table.
The beneficial effects of the invention are as follows:
1. the pymetrozine degrading bacteria IURM F18 which can survive in the environment with pymetrozine as the only carbon source is screened out, and degradation analysis shows that the bacterial strain can grow well in a solid culture medium and a liquid culture medium containing 100mg/L of pymetrozine, and has high pymetrozine degrading characteristics.
2. The pymetrozine degrading bacterium IURM F18 has application prospects in the degradation of pymetrozine in soil, the degradation of pymetrozine in water body pollution, the degradation of waste pymetrozine in soil and the restoration of environmental pollution.
3. The application of the pymetrozine degrading bacterium IURM F18 is beneficial to avoiding secondary pollution caused by physical or chemical method degradation of pymetrozine.
Drawings
FIG. 1 is a morphological diagram of a single colony of the pymetrozine-degrading bacterium IURM F18.
FIG. 2 shows the rate of degradation of pymetrozine measured by inoculating 1% of pymetrozine-degrading bacteria IURM F18 in 100mL of sterile MSM.
FIG. 3 shows the pymetrozine degradation rate measured by inoculating 1% of pymetrozine-degrading bacteria IURM F18 into 100mL of waste water containing pymetrozine.
FIG. 4 shows the rate of degradation of pymetrozine measured by inoculating 1% of the IURM F18 strain into 100g of sterilized soil.
Detailed Description
Screening process of pymetrozine degrading bacterium IURM F18
(I) main material
1. Culture medium
Pymetrozine screening liquid medium components: every 1L of deionized water contains 100mg/L of pymetrozine pesticide, 0.66g of ammonium chloride and dipotassium hydrogen phosphate (K) 2 HPO 4 ) 1.5g of potassium dihydrogen phosphate (KH) 2 PO 4 ) 0.5g magnesium sulfate (MgSO) 4 ) 0.1g of dipotassium hydrogen phosphate (K) 2 HPO 4 ) 1.5g, 0.35g of sodium chloride, and 7.0-7.2 of pH;
the corresponding solid culture medium is prepared by adding 15g of agar powder into 1L of liquid culture medium.
Composition of LB liquid Medium per 1L: separately adding 10g of peptone, 5g of yeast powder and 10g of NaCl, adjusting the pH of the prepared liquid culture medium to 7.0-7.2 by using HCl or NaOH, and then adding deionized water to complement to 1L.
The corresponding LB solid culture medium is prepared by adding 15g of agar powder into 1L of liquid culture medium.
2. Instrumentation and equipment
The kit comprises a FlexCycler PCR amplification instrument, a Tanon-3500 gel imaging system, an electrophoresis apparatus of six instruments factories in Beijing, a sterilization pot, an electronic balance, a UV1900 ultraviolet visible spectrophotometer, a constant temperature incubator, an eppendorf high-speed refrigerated centrifuge, a shaking table and a low-temperature refrigerator of Qingdao Haier group.
(II) screening process of pymetrozine-degrading bacterium IURM F18
(1) Weighing an earth separation strain from sludge polluted by pymetrozine in Changzhou Wuji area of Jiangsu, weighing 5g of sludge, adding 100mL of pymetrozine degrading bacteria IURM F18 screening liquid culture medium for enrichment, and culturing for 5-7 days in a constant temperature shaking table at 35 ℃ and 180 r/min; then 3mL of culture medium is sucked and added with a new pymetrozine degrading bacterium IURM F18 screening liquid culture medium for passage, the passage is carried out for 4-5 times, streaking separation is carried out on an LB (Langmuir-Blodgett) plate, and through separation and screening for many times, microorganisms which can survive in the culture medium taking pymetrozine as a unique carbon source are finally obtained;
the pymetrozine degrading bacteria IURM F18 screening culture medium comprises the following components: every 1L of deionized water contains 100mg/L of pymetrozine pesticide, 0.66g of ammonium chloride and dipotassium hydrogen phosphate (K) 2 HPO 4 ) 1.5g of potassium dihydrogen phosphate (KH) 2 PO 4 ) 0.5g magnesium sulfate (MgSO) 4 ) 0.1g of dipotassium hydrogen phosphate (K) 2 HPO 4 ) 1.5g, 0.35g of sodium chloride, and pH 7.0-7.2;
the LB solid medium comprises the following components: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 15g of agar powder, and the volume is made up to 1L by deionized water.
(2) Streaking and separating the culture on an LB (Langmuir-Blodgett) plate, and culturing in a constant-temperature incubator at 35 ℃ for 12-26 hours to obtain a morphological diagram of a single colony of the pymetrozine-degrading bacterium IURM F18 strain IURM F18 (shown in figure 1);
the following is a detailed description of the embodiments.
Example 1
Culturing pymetrozine-degrading bacteria IURM F18 in LB liquid culture medium, centrifugally collecting bacteria, resuspending with sterile water and diluting to OD 600 =1, inoculating 1mL of the selected strain into a basic liquid inorganic salt culture medium containing 100.0mg/L pymetrozine, placing the strain in a constant temperature shaking table for 180r/min, culturing for 30h at 35 ℃, and enabling pymetrozine degrading bacteria IURM F18 to obviously grow.
Example 2
The pymetrozine-degrading bacteria IURM F18 is inoculated into 100mL of sterile MSM according to the inoculation amount of 1%, pymetrozine is added to the final concentration of 100mg/L, the mixture is cultured for 30h under the conditions of 35 ℃ and 180r/min, and the pymetrozine degradation rate is determined to be 90.67% (see figure 2).
Example 3
Inoculating 1% of pymetrozine-degrading bacteria IURM F18 into 100mL of pymetrozine-containing wastewater, determining that the pymetrozine content of the wastewater is 58mg/L, culturing for 30h at 35 ℃ and 180r/min, and determining that the pymetrozine degradation rate is as follows: 81.23% (see FIG. 3).
Example 4
The pymetrozine-degrading bacteria IURM F18 is inoculated into 100g of sterilized soil according to the inoculation amount of 1 percent, the content of pymetrozine in the soil is determined to be 58mg/Kg, the soil is cultured for 30 hours under the constant temperature condition of 35 ℃, and the pymetrozine degradation rate is determined to be 77.01 percent (see figure 4).
Example 5
The preparation method of the dry powder of the pymetrozine-degrading bacteria IURM F18 comprises the steps of carrying out amplification culture on the pymetrozine-degrading bacteria IURM F18 and drying the products by a conventional method.
Sequence listing
<110> university of Changzhou
Jiangsu bio Environmental Protection Technology Co.,Ltd.
HEIBEI CIXIN ENVIRONMENTAL PROTECTION AND TECHNOLOGY Co.,Ltd.
<120> pymetrozine degrading bacterium IURM F18 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1425
<212> DNA
<213> sphingosine halophilum (Sphingobacterium thallophilum)
<400> 1
cagcatgggt acagtctaat acatgcaagt cggacgggat ccattggtag cttgctatca 60
atggtgagag tggagcacgg gtgcgtaacg cgtgagcaac ctgcctccat cagggggata 120
gcctctcgaa agagagatta acaccgcata acataatgtt ccggcatcgg acgattatta 180
aatatttata ggatggagat gggctcgcgt gacattagct agttggtggg gtaacggctc 240
accaaggcga cgatgtatag gggctctgag aggagaatcc cccacactgg tactgagaca 300
cggaccagac tcctacggga ggcagcagta aggaatattg gtcaatgggg ggaagcctga 360
accagccatg aagagtgcag gatgactgcc ctatgggttg taaactgctt ttgtccggga 420
ataaacctcc ttacgagtag ggagctgaat gtaccggaag aataaggatc ggctaactcc 480
gtgccagcag ccgcggtaat acggaggatc cgagcgttat ccggatttat tgggtttaaa 540
gggtgcgtag gcggccgtta agtcaggggt gaaatacggt ggctcaacca tcgcagtgcc 600
tttgatactg acgggcttga atccatatga agtgggggga ataagacaag tagcggtgaa 660
atgcatagat atgtcttaga actccgattg cgaaggcagc tcactaagct ggtattgacg 720
ctgatgcacg aaagcgtggg gatcgaacag gattagatac cctggtagtc cacgccctaa 780
acgatgataa ctcgatgttg gcgatagaca gccagcgtcc cagcgaaagc gttaagttat 840
ccacctgggg agtacgcccg caagggtgaa actcaaagga attgacgggg gcccgcacaa 900
gcggaggagc atgtggttta attcgatgat acgcgaggaa ccttacccgg gcttgaaagt 960
tcgtggagga tgcagagacg catccgtcct tcgggacacg aaactaggtg ctgcatggct 1020
gtcgtcagct cgtgccgtga ggtgttgggt taagtcccgc aacgagcgca acccctatgt 1080
ttagttgcca gcatgttatg gtggggactc taaacagact gcctgtgcaa acagtgagga 1140
aggtggggac gacgtcaagt catcatggcc cttacgtccg gggctacaca cgtgctacaa 1200
tggacggtac agcgggcagc tagctggcaa cagcatgcta atctctaaaa gccgttcaca 1260
gttcggatcg gggtctgcaa ctcgaccccg tgaagttgga ttcgctagta atcgcgtatc 1320
agcaatgacg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca agccatgaaa 1380
gttgggggta cctaaagcat gttaccgcaa ggagcggtca ggtac 1425
Claims (7)
1. A pymetrozine-degrading bacterium IURM F18 is characterized in that the pymetrozine-degrading bacterium IURM F18 is sphingomonas halophilus (Sphingobacterium halophilum), wherein the pymetrozine-degrading bacterium IURM F18 has the code: IURM F18, deposited in units of: china general microbiological culture collection center with the collection number of CGMCC No.20853.
2. The use of pymetrozine-degrading bacteria IURM F18 as claimed in claim 1, wherein said pymetrozine-degrading bacteria have a repairing effect on an environment contaminated by pymetrozine.
3. The use according to claim 2, wherein said pymetrozine-degrading bacterium IURM F18 has a repairing effect on the environment contaminated by the rejected pymetrozine in the soil.
4. The use according to claim 2, wherein the pymetrozine-degrading bacterium IURM F18 has a repairing effect on the water environment polluted by pymetrozine in water bodies.
5. The use of the IURM F18, which is an aphidone-degrading bacterium, according to claim 1, wherein the IURM F18 has a degrading effect on waste pymetrozine in soil.
6. The use of the IURM F18, which is an bacterium degrading pymetrozine, according to claim 1, wherein the IURM F18 has a degrading effect on pymetrozine contained in a water body.
7. The dry powder of the pymetrozine-degrading bacterium IURM F18 according to claim 1, which is prepared by drying the expanded-culture pymetrozine-degrading bacterium IURM F18 culture by a conventional method.
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