CN114317290B - Bacterial strain capable of degrading diuron and application thereof - Google Patents
Bacterial strain capable of degrading diuron and application thereof Download PDFInfo
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Abstract
The invention discloses a strain capable of degrading diuron and application thereof. The strain is Alternaria sp strain QHSH-5 with a collection number of CGMCC No.23258. The strain can be used for degrading pesticide diuron, is beneficial to solving the problem of agricultural diuron residue and improves the quality safety of agricultural products.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and relates to a strain capable of degrading diuron and application thereof.
Background
Diuron (Diuron) is a selective herbicide, is insoluble in water and is not soluble in most organic solvents, has stable chemical properties in air, can be decomposed when meeting strong acid and strong alkali, is not combusted, has no corrosiveness, and has the molecular weight of: 233.0945, molecular formula is: c (C) 9 H 10 Cl 2 N 2 O has the chemical structural formula as follows:
along with the fact that China becomes a large pesticide production country, the production and use of diuron in China are also rapidly increased, and diuron is applied to cotton, corn, peanut, sugarcane and fruit trees; preventing and killing annual weeds such as horse ponds, dry barnyard grass, green bristlegrass, nutgrass flatsedge, polygonum, chenopodium quinoa and the like, and has good prevention and control effects on perennial weeds such as bermuda grass, nutgrass galingale rhizome and the like. At present, the demand of diuron for sugarcane in China is about 1500 tons. Because of the large amount of diuron used, the ground water and soil have diuron residues, and although the diuron residues are low-toxicity herbicides, toxic symptoms still appear after the diuron is taken by mistake, the diuron residues have a stimulating effect on the upper respiratory tract, and the minimum lethal dose of human mouth is 500mg/kg. Diuron has been shown to be an endocrine disrupter and can have adverse effects on humans after contact.
In the use of pesticides, most of pesticides enter air, water and soil, and a large amount of pesticide residues are caused in the water and soil because only a small part of pesticides volatilize. In the current pesticide residue degradation method, compared with the traditional degradation method, the microbial degradation has unique advantages, can carry out bioremediation and decomposition on the pesticide, converts the pesticide into simple inorganic matters, and has the characteristics of safety, high efficiency, rapidness, low cost and no secondary pollution. And the strain with degradation effect can be separated and screened out, which is favorable for solving the problem of pesticide residue, improving the quality of agricultural products and promoting the agricultural development.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a strain with degradation effect on diuron. The strain can effectively degrade pesticide diuron, lays a foundation for solving the problem of diuron residue, and can improve the environment of soil, water and the like.
To achieve the above object, the present invention provides Alternaria sp strain QHSH-5 having a collection number of CGMCC No.23258.
The strain QHSH-5 is characterized in that commercial Martin fungus culture medium is cultured for 3 days at 28 ℃, the colony form is round, and the strain is easy to pick after drying, opaque, villous and has white hypha on the surface.
The Alternaria sp. Strain QHSH-5 of the present invention was selected as follows,
1. isolation and purification and screening of strains
Separating and purifying a single microorganism strain from diuron polluted soil by means of gradient dilution, plate scribing and the like, and using the single microorganism strain for subsequent experiments.
The single microorganism strains are respectively inoculated into a culture medium, and shake culture is carried out in a shaking table at 28 ℃ and 180r/min, which is an experimental group, and a self control group and a standard control group are arranged. When the strain grows to the logarithmic growth phase, diuron is added into the experimental group and the standard control group, the concentration is 50mg/L, the temperature is 28 ℃, the samples are respectively sampled at 180r/min for thin-layer analysis to observe the degradation effect of the strain on pesticides, and the strain with the degradation effect is selected as QHSH-5 and used as a next study object.
Identification of QHSH-5
(1) Morphological identification
The strain QHSH-5 was inoculated onto a commercial Martin solid plate and cultured at 28℃for 3d, and the colony morphology was observed. The colony forms a round shape, is dry and opaque, and has white hypha on the villus surface (see figure 1).
(2) Molecular biological identification
Extracting genome DNA of the strain, and carrying out PCR amplification and sequencing by taking the extracted genome as a template. The ITS sequences measured by the strain are compared and analyzed in NCBI database by BLAST, and related sequences with higher homology are selected to construct phylogenetic tree and analyze evolutionary relationship by MAGE 7.0 software (see figure 2).
Finally, QHSH-5 was identified as Alternaria sp according to morphological characteristics, homology, phylogenetic tree, and literature report.
Degradation assay of QHSH-5
(1) Growth curve determination
The purified strain QHSH-5 was inoculated in a commercial Martin fungus medium, 3 groups were parallel, and the growth curves were measured by dry weight method after sampling for 2, 4, 6, 8, 10 days of culture, respectively (see FIG. 3).
(2) Isolation of the conversion products
The strain QHSH-5 is fermented in batches, diuron is added in the logarithmic growth phase, mycelia are removed by filtration until the 10 th day, supernatant is concentrated after passing through a macroporous resin column, then is separated by column chromatography, and a sample is selected to further determine the degradation product structure of the QHSH-5 on the diuron through nuclear magnetic resonance hydrogen spectrum and carbon spectrum (see figures 4 and 5).
(3) Degradation assay
The degradation efficiency of diuron by diuron degrading bacteria QHSH-5 was determined by adding diuron to strain QHSH-5 and sampling at regular time, then performing HPLC-DAD measurement, and measuring the content of diuron and degradation products (see FIGS. 6, 7 and 8).
The invention also provides application of the strain or the culture solution thereof or the fermentation solution thereof or the microbial inoculum containing the strain or the fermentation solution thereof in degradation of diuron.
The invention also provides application of the strain or the culture solution thereof or the fermentation solution thereof or the microbial inoculum containing the strain or the fermentation solution thereof in preparation of diuron degradation products.
The invention also provides application of the strain or the culture solution thereof or the fermentation liquor thereof or the microbial inoculum containing the strain or the fermentation liquor thereof in reducing the residue of diuron after use.
The invention also provides a diuron degradation product, which comprises the strain or the culture solution or the fermentation solution or the microbial inoculum containing the diuron degradation product.
The Alternaria sp.QHSH-5 with diuron degradation effect is screened out, can be used for degrading pesticide diuron, is beneficial to solving the problem of agricultural diuron residue and improves the quality safety of agricultural products.
Preservation description
Strain name: alternaria alternata (L.) Kuntze
Latin name: alternaria sp.
Strain number: QHSH-5
Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
The preservation organization is abbreviated as: CGMCC
Address: beijing city, chaoyang area, north Chenxi Lu No.1 and 3
Preservation date: 2021, 9, 24
Accession numbers of the preservation center: CGMCC No.23258
Drawings
FIG. 1 is a graph of morphology features of individual colonies of QHSH-5.
FIG. 2 is an ITS phylogenetic tree of QHSH-5.
FIG. 3 is a graph of the growth curve of QHSH-5.
FIG. 4 is a nuclear magnetic resonance spectrum of diuron degradation products.
FIG. 5 is a nuclear magnetic resonance spectrum of diuron degradation products.
FIG. 6 shows the results of QHSH-5 degradation experiments on diuron.
FIG. 7 is a liquid phase diagram of diuron in QHSH-5 degradation experiments.
FIG. 8 is a liquid phase diagram of diuron degradation products in QHSH-5 diuron degradation experiments.
FIG. 9 is a thin layer chromatogram of the negative results of screening different strains for diuron degradation activity.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The medium formulation in the following examples is as follows:
commercial martin medium: 5.0g of peptone, 1.0g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate, 2.0g of yeast extract powder, 20.0g of glucose, 1000mL of distilled water, pH=7.0 and sterilization at 121 ℃ for 20min. Solid medium: 15.0g of agar powder is added per 1L.
Example 1 isolation, purification and screening of Strain QHSH-5
1. Isolation and purification of strains
Collecting diuron contaminated soil from PingLUO county in Ningxia Shizui mountain city 7 months 2020, mixing 2g in 50mL sterile water thoroughly, standing and clarifying, and respectively diluting the supernatant to 10 by gradient dilution method -1 、10 -2 、10 -3 、10 -4 、10 -5 Uniformly coating the mixture in a commercial Martin solid culture medium in an equal gradient, culturing at 28 ℃, and purifying by a plate streak separation method to obtain a plurality of single purified microorganism strains.
2. Screening of diuron degrading bacteria
Commercial Martin liquid medium (without agar added) was prepared, 150mL of medium was contained in a 250mL Erlenmeyer flask, and a single purified plurality of strains was inoculated into the medium, and shaking culture was performed at 28℃and 180 r/min. After the strain grows to the logarithmic phase, adding a substrate diuron with the concentration of 50mg/L, continuously culturing for 5 days, 7 days and 9 days under the same condition, respectively sampling, carrying out thin-layer detection, and analyzing the result.
Standard control group was established: diuron (50 mg/L) was added to an equal amount of liquid medium;
setting up a self control group: the above strains were inoculated into an equal amount of liquid medium in the same manner as above, but diuron was not added.
The thin layer analysis result is influenced by the self secondary metabolism interference of bacteria removed by the self control group; interference of the standard control discharge medium on the compound diuron. According to the thin-layer information, the degradation of different bacteria to pesticide diuron is observed, and the strain with obvious degradation effect is selected as a next research object. A strain with degradation effect on diuron is obtained, and the number is QHSH-5.
In the QHSH-5 obtaining process, a large number of experiments are carried out, and the strains for screening are nearly 200 strains (the analysis result of partial screening thin layer is shown in figure 9), and also comprise other strains which are the same as Alternaria alternata, such as Alternaria alternata CGMCC No.15984 obtained in the earlier stage of the laboratory, however, only one active strain is screened out, which proves that diuron is difficult to degrade, and is also the reason that diuron pesticide residues in soil are serious.
Example 2 identification of Strain QHSH-5
(1) Colony morphology
QHSH-5 was inoculated onto commercial Martin medium plates, and the colony morphology was round, dried, opaque, fluffy, and had white hyphae on the surface. The gray hyphae germinate at the initial stage and then spread circularly to the culture medium, and the middle color of the medium becomes gradually darker around the gray hyphae after rounding. (see FIG. 1)
(2) Identification of strains
QHSH-5 is inoculated into a liquid commercial Martin culture medium, the temperature is 28 ℃, the speed is 180r/min, shaking culture is carried out for 3d, and a small amount of thalli is taken after centrifugation. QHSH-5 genomic DNA was extracted using a "fungus genomic DNA extraction kit" (product number: B004009020, tin-free Baitaike Biotechnology Co., ltd.) and the extraction procedure was referred to the fungus genomic DNA extraction kit instructions. PCR amplification was performed using QHSH-5 genomic DNA as template and primers ITS1 and ITS 4.
The sequence of the fungal primer is selected as follows:
ITS1(5′-TCCGTAGGTGAACCTGCGG-3′)
ITS4(5′-TCCTCCGCTTATTGATATGC-3′)
the PCR amplification reaction system (50. Mu.L) included: 5×buffer 10. Mu.L; dNTP 4. Mu.L; 1. Mu.L of forward primer (10. Mu.M); reverse primer (10. Mu.M) 1. Mu.L; prime STAR 0.5 μL; template (1. Mu.M) 1. Mu.L; ddH 2 O 32.5μL。
PCR reaction conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 2min,30 cycles; extending at 72℃for 5min.
The amplified product (i.e., ITS of QHSH-5) was submitted to Beijing Yougi technologies Co., ltd for sequencing.
The ITS sequence of QHSH-5 is shown as SEQ ID No.1, the obtained sequence is subjected to homology comparison analysis with NCBI nucleic acid database by BLAST, a strain with high partial similarity is selected to carry out phylogenetic analysis with the QHSH-5 strain, and a phylogenetic tree is constructed by using a Neighbor-Joining method (Neighbor-Joining) in MEGA 7.0 software. Phylogenetic tree is shown in fig. 2.
Finally, QHSH-5 was identified as Alternaria sp according to morphological characteristics, homology, phylogenetic tree, and literature report.
EXAMPLE 3 degradation assay of degrading bacterium QHSH-5
(1) Growth curve determination
Taking an inclined plane preservation test tube for preserving degrading bacteria QHSH-5, adding 3mL of liquid commercial Martin culture medium, repeatedly blowing and sucking, washing off fungus fur to obtain mycelium eluent, transferring the mycelium eluent to another sterile test tube by using a suction tube, diluting by 10 times with sterile water, and preparing 10mL of fungus suspension. 15 bottles of liquid commercial Martin medium were prepared, 150mL of liquid commercial Martin medium was contained in each 250mL conical flask, and each flask was inoculated with 0.5mL of QHSH-5 bacterial suspension and cultured at a constant temperature of 28℃and 180r/min by shaking. Set 3 groups in parallel, sample for 2, 3, 4, 5, 6, 7, 8, 9 days, retain mycelium, dry to constant weight and weigh, and measure growth curve by dry weight method (see figure 3). As shown in FIG. 3, the growth curve is the logarithmic growth phase for the first 6 days, the maximum value is reached on the 6 th day, and the decay phase is reached after the 6 th day, so that the optimum culture time of QHSH-5 is 6 days.
(2) Separation of degradation products
Carrying out batch fermentation on diuron degrading bacteria QHSH-5, preparing 34 bottles of liquid commercial Martin culture medium, wherein each conical flask contains 150mL of culture medium, and carrying out constant-temperature shaking culture on the diuron degrading bacteria QHSH-5 at the temperature of 28 ℃ and the constant temperature of 180 r/min. 500mg diuron is dissolved in 6.8ml DMSO, 200 mu L of diuron is added to each bottle of culture medium, i.e. 500mg diuron is added in the logarithmic growth phase, and mycelium is removed by filtration until the 10 th day to obtain fermentation broth. The fermentation liquor is passed through D101 macroporous adsorption resin chromatographic column (phi 75mm multiplied by 150 cm), washed with water, washed with 95% ethanol, then the sample is concentrated, the concentrated sample is subjected to silica gel mixing, then is subjected to silica gel column chromatography (phi 26mm multiplied by 457 mm) to separate (dichloromethane/methanol gradient elution), a crude sample containing diuron degradation product is obtained according to thin layer analysis, and then the crude sample is passed through Sephadex LH-20 gel column (phi 17mm multiplied by 457 mm) (petroleum ether/dichloromethane/methanol 5:4:1 elution) to obtain degradation product of QHSH-5 to diuron, and the structural formula of the degradation product of QHSH-5 to diuron is determined through nuclear magnetic resonance hydrogen spectrum and carbon spectrum (see figure 4 and figure 5) as shown below, and is diuron demethylated product.
The diuron demethyl product spectrum data were attributed as follows:
1 H-NMR(MeOH-d 4 ，400MH)：7.72(d，J＝2.8Hz，H-2)，7.35(d，J＝8.8Hz，H-5)
7.21(dd，J＝8.8，2.8Hz，H-6)，2.76(s，H CH3 -10)；
13 C-NMR(MeOH-d 4 ，100MH)：158.5(C-8)，141.5(C-1)，133.4(C-3)，131.5(C-4)，125.7(C-5)，121.2(C-6)，119.4(C-2)，26.9(C-10)。
d101 macroporous adsorbent resin (Beijing Soy Bao technology Co., ltd.; M0041) is selected, and silica gel is laminated on a column layer of 200-300 meshes (Qingdao ocean chemical Co., ltd.; M150107), and Sephadex LH-20 (GE Healthcare Life Sciences China; 17009002).
(3) Degradation rate measurement
Washing with sterile physiological saline to preserve the lawn in the slant preservation test tube of the degrading bacteria QHSH-5, mixing thoroughly, diluting with physiological saline to 10 -2 Doubling to obtain bacterial suspension of QHSH-5 with concentration of 10 6 CFU/mL. The treatment group is inoculated in a liquid commercial Martin culture medium with an inoculum size of 10 percent, 3 groups are arranged in parallel, the culture is carried out at the constant temperature of 28 ℃ and 180r/min, 150mg/L diuron is added in the logarithmic phase of culture, sampling is carried out on days 2, 4, 6, 8 and 10 after diuron is added, the degradation amount in the diuron degradation process is dynamically monitored, the diuron and degradation products in the culture solution are detected by adopting HPLC-DAD, the liquid commercial Martin culture medium under the same condition is not inoculated with strains as a control group, and diuron standard (Shanghai Ala Biotechnology Co., ltd., D101260) and the peak time of the separated degradation products are used as controls to judge the diuron and the degradation products in the culture solution (see figures 7 and 8). Fig. 7 is a liquid chromatogram of diuron, and fig. 8 is a liquid chromatogram of degradation products.
Determination of diuron degradation rate and yield of degradation products: centrifuging the culture solution to remove thallus, collecting supernatant 20mL, and passing through ODS C 18 And (3) a small column, reserving a methanol layer, dissolving with chromatographic pure methanol after volatilizing, filtering by using a 0.45 mu m organic filter membrane, and placing in a brown chromatographic bottle to obtain a sample to be detected.
The analysis adopts a high performance liquid chromatograph: shimadzu LC2030C chromatograph, megers C18 column (250×4.6mm,10 μm), mobile phase methanol: water = 60:40, the flow rate is 1mL/min, the column temperature is 30 ℃, the sample injection amount is 10 mu L, and the detection wavelength is 254nm.
The degradation experiment results are shown in fig. 6, and the degradation rate (%) = (diuron concentration in control group-diuron concentration in treatment group)/diuron concentration in control group is multiplied by 100%.
The diuron content tended to decrease rapidly over time and showed a very high degradation rate of QHSH-5 to diuron, about 74%, based on the continuous increase in degradation product content over time.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Sequence listing
<110> university of Ningxia medical science
Ningxia national academy of sciences of agriculture and forestry plant protection institute (Ningxia plant pest control key laboratory)
<120> a strain capable of degrading diuron and use thereof
<130> GNCYZ220254
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 573
<212> DNA
<213> Alternaria sp.)
<400> 1
gtggctgcca tgaggcgggc tggaaccctc cagccgggca ctgcttcacg gcgtgcgcgg 60
ctggagccgg ccctgctgaa ttattcaccc gtgtcttttg cgtacttctt gtttcctggg 120
tgggctcgcc cgccatcagg accaaccata aacctttttg taatagcaat cagcgtcagt 180
aacaacgtaa ttaattacaa ctttcaacaa cggatctctt ggttctggca tcgatgaaga 240
acgcagcgaa atgcgatacg tagtgtgaat tgcagaattc agtgaatcat cgaatctttg 300
aacgcacatt gcgccctttg gtattccaaa gggcatgcct gttcgagcgt catttgtacc 360
ctcaagcttt gcttggtgtt gggcgtcttt tgtctccagc tcgctggaga ctcgccttaa 420
agtcattggc agccggccta ctggtttcgg agcgcagcac aagtcgcgct cttgcccagc 480
caaggtcagc gtccagcaag ccttttttca acctttgacc tcggatcagg tagggatacc 540
cgctgaactt aagcatatca ataagcggag gaa 573
Claims (5)
1. Alternaria genusAlternaria sp.) The preservation number of the strain QHSH-5 is CGMCC No.23258.
2. Use of the strain of claim 1 or a fermentation broth thereof or a microbial inoculum containing the same for the degradation of diuron.
3. Use of the strain of claim 1 or a fermentation broth thereof or a microbial inoculum containing the same for the preparation of a degraded diuron product.
4. Use of a strain according to claim 1 or a fermentation broth or a microbial inoculum containing the strain for reducing the residual quantity of diuron after use.
5. The diuron degrading product is characterized in that: a strain comprising the strain of claim 1 or a fermentation broth thereof or a microbial inoculum comprising the same.
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