CN114231457A - Bacillus cereus capable of rapidly degrading various pesticide residues - Google Patents

Abstract

The invention discloses a broad-spectrum degradation strain for various pesticide residues, which is identified as bacillus cereus 22-2 (B)Bacillus cereus 22-2) and has been preserved in China center for type culture Collection with the preservation time of 26 months at 2021 and the preservation number of CCTCC NO: m2021936. The bacillus cereus 22-2 provided by the invention is applied to soil and canSo as to effectively degrade various pesticide residues, reduce the pollution of chemical pesticides to soil and reduce the pesticide residues of crops; meanwhile, the pesticide resistance of pathogenic bacteria caused by the action of chemical pesticides is effectively reduced, the balance of a soil micro-ecosystem is protected, the living environment of crops is improved, the disease resistance of the crops is improved, and the method has important significance for increasing the yield and improving the quality of the crops.

Description

Bacillus cereus capable of rapidly degrading various pesticide residues

Technical Field

The invention relates to the technical field of microorganisms, in particular to bacillus cereus capable of rapidly degrading various pesticide residues.

Background

The national soil pollution condition survey bulletin jointly issued by the ecological environment department and the natural resource department of China in 2014, 4, 17 shows that the organic pollution is serious in the soil of China at present, and the overproof condition of organic pollutants is second to the heavy metal pollution. The pesticide in the organic pollutants is one of important pollution sources, has the properties of high toxicity, high residue and difficult degradation, is an important agricultural chemical, is widely used all over the world, and plays an important role in promoting agricultural development in the aspects of preventing and treating agricultural plant diseases and insect pests and ensuring the yield and income increase of crops. Since the birth of agricultural chemicals, the amount of agricultural chemicals used is increasing worldwide in order to meet the demand for food by population growth. China is a big agricultural country, a large amount of pesticides are used every year for preventing and treating crop diseases and insect pests, and the annual average usage amount and export amount of the pesticides are at the top of the world. The pesticide can cause certain threat to the health of agricultural workers in the using process, and causes pollution to ecological environments such as biological communities, soil, water bodies, atmosphere and the like and even global ecosphere. In order to solve this conflict, it is necessary to develop a highly effective, low-toxic, low-residue chemical pesticide or biopesticide, and to find a preparation capable of degrading the pesticide residue efficiently and rapidly.

Bioremediation technology is a biological engineering technology for eliminating and treating environmental pollution appeared and developed in the 80 s, and the bioremediation technology comprises phytoremediation, animal remediation, microbial remediation, biological combined remediation and the like. Theoretically, the technical route has the greatest potential and is expected to realize the most thorough repairing effect.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: provides a bacillus cereus capable of rapidly degrading various pesticide residues.

In order to solve the technical problems, the invention adopts the technical scheme that:

the invention provides a pesticide residue broad-spectrum degradation strain which is identified as Bacillus cereus 22-2(Bacillus cereus 22-2), is preserved in China center for type culture collection, has the preservation time of 2021, 7 months and 26 days, and has the preservation number of CCTCC NO: m2021936.

The strain is obtained by screening greenhouse soil with 20 years of vegetable planting history in Shandong, and has rapid degradation effect on imidacloprid, acetamiprid, triazolone, fosthiazate, chlorothalonil, cyhalothrin and other pesticides, wherein the pesticides capable of being efficiently and rapidly degraded are acetamiprid, fosthiazate and triazolone.

The strain is characterized in that the strain can form a round or approximately round white candle-like colony with soft texture, no pigment and slight luster on an LB plate culture medium at 30 ℃ for 48 hours, the diameter is 5-7mm, the culture time is slightly long, and the surface and the side surface of the colony are in a ground glass shape.

The bacterial strain is subjected to molecular biological identification, a 16S rDNA sequence is measured, Blast comparison is carried out in a GenBank nucleic acid database, and the bacterial strain is confirmed to be Bacillus cereus (Bacillus cereus) and named as Bacillus cereus 22-2(Bacillus cereus 22-2) by combining the biological characteristics of the bacterial strain and the 16S rDNA comparison result.

The invention also provides application of the bacillus cereus 22-2 in degrading imidacloprid, acetamiprid, triazolone, fosthiazate, chlorothalonil and cyhalothrin pesticide residues.

Further, the bacillus cereus 22-2 can degrade pesticide residues such as imidacloprid, acetamiprid, triazolone, fosthiazate, chlorothalonil, cyhalothrin and the like in soil or water body environment, wherein the acetamiprid, the fosthiazate and the triazolone can be degraded quickly and efficiently.

The invention also provides a pesticide residue degrading microbial inoculum produced by using the bacillus cereus 22-2. The pesticide residue degrading microbial inoculum has a rapid degrading effect on pesticides such as acetamiprid, fosthiazate and triadimefon which pollute soil.

The invention also provides a preparation method of the pesticide residue degrading microbial inoculum, which comprises the following steps:

(1) inoculating the test tube liquid of bacillus cereus 22-2 cultured to logarithmic phase into a fermentation culture medium according to 0.5-1% of the volume of the fermentation culture medium, and culturing to logarithmic phase to obtain a fermentation strain;

(2) inoculating the prepared fermentation strain into a culture medium of a seed tank according to 5-10% of the volume of the culture medium of the seed tank, and culturing to logarithmic phase to prepare a seed solution;

(3) inoculating the seed liquid into the culture medium of a production tank according to 5-10% of the volume of the culture medium of a 5 ton production tank, culturing and fermenting, wherein the fermentation temperature is 32 ℃, the fermentation is stopped when the microscopic spore rate reaches more than 95%, and after the fermentation is finished, light calcium powder with the mass of 12.2% is added, and after centrifugation, spray drying is carried out to prepare a powder product with the bacterial load of 10 hundred million/g.

Furthermore, the invention also further performs molecular biological detection on the strain to provide gene islands, GO function, KEGG function, COG function, NR function and TCDB function genes and the quantity of the bacillus cereus 22-2.

The invention has the beneficial effects that:

the bacillus cereus 22-2 provided by the invention is applied to soil, can effectively degrade various pesticide residues, efficiently degrade acetamiprid, fosthiazate and triazolone pesticides, reduce the pollution of chemical pesticides to the soil and reduce the pesticide residues of crops; meanwhile, the pesticide resistance of pathogenic bacteria caused by the action of chemical pesticides is effectively reduced, the balance of a soil micro-ecosystem is protected, the living environment of crops is improved, the disease resistance of the crops is improved, and the method has important significance for increasing the yield and improving the quality of the crops.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is a statistical plot of the gene distribution in the Bacillus cereus 22-2 gene island. The strain is subjected to frame sequencing, IslandPath-DIOMB software (Version 0.2) is adopted to predict gene islands, and the sequencing result shows that the strain has 14 gene islands.

FIG. 2 Bacillus cereus strain 22-2 Gene functional Annotation GO functional taxonomic map. For Gene function annotation of the strain 22-2, the general name of GO function is Gene Ontology, and the GO function is a set of international standardized classification systems for Gene function description. GO is divided into three major classes: 1) cell fraction (Cellular Component): for describing subcellular structures, locations, and macromolecular complexes, such as nucleoli, telomeres, and complexes that recognize initiation; 2) molecular Function (Molecular Function): for describing the functions of the gene, individual gene products, such as carbohydrate binding or ATP hydrolase activity, etc.; 3) biological Process (Biological Process): is used to describe the biological processes involved in the gene-encoded product, such as mitosis or purine metabolism. The statistical results of the three major taxonomic genes in the GO database are shown in the following chart. Description of the drawings: the abscissa indicates GO functional classification on sample annotation, the right ordinate indicates number of genes on annotation, and the left ordinate indicates percentage of number of genes on annotation to all encoded genes.

FIG. 3 Bacillus cereus 22-2 gene function annotation KEGG metabolic pathway classification map. KEGG is known as Kyoto Encyclopedia of Genes and genomics. Databases of metabolic pathways of gene products and compounds in cells and the function of these gene products are systematically analyzed. It integrates data in GENOME, chemical molecule and biochemical systems, including metabolic pathway (KEGG PATHWAY), DRUG (KEGG DRUG), disease (KEGG DISEASE), functional model (KEGG MODULE), gene sequence (KEGG GENES) and GENOME (KEGG GENOME), etc. The KO (KEGG ORTHOLOG) system connects various KEGG annotation systems together, and the KEGG establishes a complete set of KO annotation systems which can complete the functional annotation of the genome or transcriptome of a new sequencing species. Description of the drawings: the numbers on the bar represent the number of genes on the annotation; the other coordinate axis is the code of each function class of level1 in the database.

FIG. 4 functional annotation COG functional taxonomy map of the Bacillus cereus 22-2 gene. COG is called Cluster of organizations of proteins, and is constructed by the protein database created and maintained by NCBI and classified according to the phylogenetic relationship of the encoded proteins of the complete genomes of bacteria, algae and eukaryotes. A certain protein sequence can be annotated to a certain COG by alignment, and each cluster of COG is composed of an orthologous sequence, so that the function of the sequence can be presumed. COG databases can be divided into twenty-six categories according to function. The abscissa indicates the COG function type, and the ordinate indicates the number of genes annotated.

FIG. 5 functional annotation of the NR functional taxonomy map for the Bacillus cereus 22-2 gene. NR is called Non-Redundant Protein Database, is a Non-Redundant Protein Database, is created and maintained by NCBI, and is characterized in that the content is relatively comprehensive, and meanwhile, the annotation result contains species information which can be used for species classification. According to the annotated species of the gene, the annotated species and the number of the gene are counted, and the statistical result is shown in the following chart. The abscissa represents the species ID and the ordinate represents the number of genes annotated.

FIG. 6 functional Annotation TCDB functional Classification of Bacillus cereus 22-2 genes. The CDB is called Transporter Classification Database, Transporter taxonomy Database, and is a membrane Transporter, including the sorting system (TC system) of ion channels (ion channels). The TCDB database migration system is classified in 5 levels, and the first level statistics are as follows. The abscissa represents the TCDB class one type of classification, and the ordinate represents the number of genes annotated.

Detailed Description

Example 1 Strain Source and identification

1. The strain source is as follows:

the embodiment provides bacillus cereus which is obtained by screening greenhouse soil with 20-year vegetable planting history in Shandong Shouguang, and has degradation effects on imidacloprid, acetamiprid, triazolone, fosthiazate, chlorothalonil and cyhalothrin, wherein the bacillus cereus has rapid and efficient degradation effects on acetamiprid, fosthiazate and triazolone pesticides remained in the soil.

2. Is characterized in that:

culturing on LB plate culture medium at 30 deg.C for 48 hr to form round or nearly round, soft, non-pigment, slightly glossy white candle-like colony with diameter of 5-7mm, and slightly long culture time, wherein the colony surface and side surface are in ground glass shape.

The bacterial strain is subjected to molecular biological identification, a 16S rDNA sequence is measured, Blast comparison is carried out in a GenBank nucleic acid database, and the bacterial strain is confirmed to be Bacillus cereus (Bacillus cereus) and named as Bacillus cereus 22-2(Bacillus cereus 22-2) by combining the biological characteristics of the bacterial strain and the 16S rDNA comparison result. The applicant has deposited the Bacillus cereus 22-2(Bacillus cereus 22-2) in China center for type culture Collection of Wuhan university in China at 26/7/2021 with the preservation number of CCTCC NO: m2021936.

Example 2 microbial inoculum sample preparation

The embodiment provides a pesticide residue degradation microbial inoculum of bacillus cereus 22-2, and the specific method comprises the following steps:

(1) inoculating the test tube liquid of bacillus cereus 22-2 cultured to logarithmic phase into a fermentation culture medium according to 0.5-1% of the volume of the fermentation culture medium, and culturing to logarithmic phase to obtain a fermentation strain;

(2) inoculating the prepared fermentation strain into a culture medium of a seed tank according to 5-10% of the volume of the culture medium of the seed tank, and culturing to logarithmic phase to prepare a seed solution;

(3) inoculating the seed liquid into the culture medium of a production tank according to 5-10% of the volume of the culture medium of a 5 ton production tank, culturing and fermenting, wherein the fermentation temperature is 32 ℃, the fermentation is stopped when the microscopic spore rate reaches more than 95%, and after the fermentation is finished, light calcium powder with the mass of 12.2% is added, and after centrifugation, spray drying is carried out to prepare a powder product with the bacterial load of 10 hundred million/g.

Example 3 evaluation of Imidacloprid degradation ability of Bacillus cereus 22-2

1. Experiment site Shandong province goddess Weifang science and technology institute Beiluo experiment base

2. Design of experiments

The experimental field is irrigated before 2 days, the imidacloprid pesticide (98% of original drug) is diluted to 500mg/ml after two days, 100ml is uniformly applied to the circumference of the roots of the melon plants (melon plants planted for a week), 100ml of suspension containing 10g of microbial inoculum is applied at the same time, and a positive control (100 ml of imidacloprid pesticide with the concentration of 500mg/ml and 100ml of clear water are applied to the circumference of the roots) and a negative control (200 ml of clear water is applied to the circumference of the roots) are set.

3. Determination of imidacloprid pesticide degradation effect

After the polluted soil is repaired for 7 days and 9 days, soil samples within a depth range of 15cm around roots are respectively taken, the content of imidacloprid in the soil samples is measured by using a liquid chromatography-triple quadrupole mass spectrometer, meanwhile, a soil addition recovery experiment of an imidacloprid standard is carried out, the detection results are shown in the table 1-2, the recovery rate of the imidacloprid from the soil is more than 94%, and the 22-2 strain is repaired for 7 days and 9 days, and the degradation rate of the imidacloprid is respectively increased by 16.9% and 35.4% compared with the unrepaired soil.

TABLE 1 Imidacloprid-contaminated soil Bacillus cereus 22-2 degradation 7 days determination results

TABLE 2 Imidacloprid-contaminated soil Bacillus cereus 22-2 degradation 9 days determination results

Example 4 evaluation of the ability of Bacillus cereus 22-2 to degrade acetamiprid

1. Experiment site Shandong province goddess Weifang science and technology institute Beiluo experiment base

2. Design of experiments

The experimental plot is irrigated before 2 days, the acetamiprid pesticide (98% of original drug) is diluted to 500mg/ml after two days, 100ml is uniformly applied to the circumference of the roots of the melon plants (melon plants planted for a week), 100ml of suspension containing 10g of microbial inoculum is applied at the same time, and a positive control (100 ml of acetamiprid pesticide with the concentration of 500mg/ml and 100ml of clear water are applied to the circumference of the roots) and a negative control (200 ml of clear water is applied to the circumference of the roots) are set.

3. Determination of degradation effect of acetamiprid pesticide

After the contaminated soil is treated for 7 days and 14 days, soil samples within a depth range of 15cm around roots are respectively taken, the content of the acetamiprid in the soil samples is determined by using a liquid chromatography-triple quadrupole mass spectrometer, meanwhile, soil addition recovery experiments of the acetamiprid standard substance are carried out, the detection results are shown in tables 3-4, the recovery rate of the acetamiprid from the soil is more than 96%, and the degradation rates of the acetamiprid in the soil after the 22-2 strain is repaired for 7 days and 14 days are respectively increased by 45.9% and 89.8% compared with the degradation rate of the acetamiprid in the soil without repair.

TABLE 3 acetamiprid-contaminated soil Bacillus cereus 22-2 degradation 7 days determination results

TABLE 4 acetamiprid-contaminated soil bacillus cereus 22-2 degradation 14 days determination results

Example 5 evaluation of ability of Bacillus cereus 22-2 to degrade Thiazolophosphine

1. Experiment site Shandong province goddess Weifang science and technology institute Beiluo experiment base

2. Design of experiments

The experimental plot is irrigated before 2 days, the fosthiazate pesticide (98% of original drug) is diluted to 500mg/ml after two days, 100ml of the suspension containing 10g of the fungicide is uniformly applied to the circumference of the roots of the muskmelon plants (the muskmelon plants are planted for one week), and a positive control (100 ml of the fosthiazate pesticide with the concentration of 500mg/ml and 100ml of clear water are applied to the circumference of the roots) and a negative control (200 ml of clear water is applied to the circumference of the roots) are set.

3. Determination of degradation effect of fosthiazate pesticide

After the polluted soil is treated for 9 days, a soil sample with the depth range of 15cm around the root is taken, the content of the fosthiazate in the soil sample is determined by using a liquid chromatography-triple quadrupole mass spectrometer, meanwhile, a soil addition recovery experiment of a fosthiazate standard is carried out, the detection result is shown in table 5, the recovery rate of the fosthiazate from the soil is more than 97%, and the 22-2 strain recovery 9 days is increased by 76.1% compared with the unrepaired soil fosthiazate degradation rate.

TABLE 5 results of 9-day degradation of Bacillus cereus 22-2 in fosthiazate-contaminated soil

Example 6 evaluation of ability of Bacillus cereus 22-2 to degrade triazolone

1. Experiment site Shandong province goddess Weifang science and technology institute Beiluo experiment base

2. Design of experiments

The experimental field is irrigated before 2 days, the triazolone pesticide (98% of original drug) is diluted to 500mg/ml after two days, 100ml is uniformly applied to the root circumference of a melon plant (melon seedling plant planted for one week), 100ml of suspension containing 10g of microbial inoculum is applied at the same time, and a positive control (100 ml of triazolone pesticide with the concentration of 500mg/ml and 100ml of clear water are applied to the root circumference) and a negative control (200 ml of clear water is applied to the root circumference) are set.

3. Determination of degradation effect of triazolone pesticide

After the contaminated soil is treated for 7 days and 14 days, soil samples with the depth range of 15cm around roots are respectively taken, the content of the triazolone in the soil samples is measured by using a liquid chromatogram-triple quadrupole mass spectrometer, meanwhile, soil addition recovery experiments of the triazolone standard pesticide are carried out, the detection results are shown in the table 6-7, the recovery rate of the triazolone from the soil is more than 99%, and the degradation rate of the triazolone is increased by 65.5% and 66.1% compared with that of the triazolone in unrepaired soil after the 22-2 bacterial strains are repaired for 7 days and 14 days.

TABLE 6 results of 7-day degradation assay of triazolone-contaminated soil bacillus cereus 22-2

TABLE 7 results of 14-day degradation assay of triazolone-contaminated soil bacillus cereus 22-2

Example 7 evaluation of the ability of Bacillus cereus 22-2 to degrade chlorothalonil

1. Test site: shaoyuang city Weifang science and technology institute Beiluo experimental base of Shandong province

2. Design of experiments

The experimental plot is irrigated before 2 days, after two days, chlorothalonil pesticide (98% of original drug) is diluted to 1000mg/ml, 100ml is uniformly applied to the circumference of roots of melon plants (melon plants planted for a week), 100ml of suspension containing 10g of microbial inoculum is simultaneously applied, and positive control (100 ml of chlorothalonil pesticide with the concentration of 500mg/ml and 100ml of clear water are applied to the circumference of the roots) and negative control (200 ml of clear water is applied to the circumference of the roots) are set.

3. Determination of degradation effect of chlorothalonil pesticide

The method comprises the steps of treating the contaminated soil for 9 days and 14 days, taking soil samples within a depth range of 15cm around roots, measuring the content of chlorothalonil in the soil samples by using a gas chromatography-mass spectrometer, and simultaneously performing a soil addition recovery experiment on a chlorothalonil standard product, wherein the detection results are shown in tables 8-9, the recovery rate of the chlorothalonil from the soil is over 90 percent, and the 22-2 strain recovery 9 days is increased by 87.1 percent and 93.8 percent compared with the unrepaired soil chlorothalonil.

TABLE 8 determination of Bacillus cereus 22-2 degradation in chlorothalonil contaminated soil for 9 days

TABLE 9 determination of Bacillus cereus 22-2 degradation in chlorothalonil contaminated soil for 14 days

Example 8 Gene annotation of Bacillus cereus 22-2 Strain

Extracting genome DNA of the bacillus cereus 22-2, utilizing random PCR amplification and sequence double-end sequencing to obtain original Data (Raw Data) obtained by sequencing, wherein the original Data can have a certain proportion of low-quality Data, and in order to ensure the accuracy and reliability of a subsequent information analysis result, firstly, filtering the original Data to obtain effective Data (clear Data), and the steps are as follows:

(1) removing reads containing low-quality bases (with the quality value less than or equal to 20) in a certain proportion (default set as 40%);

(2) removing N bases to reach a certain proportion of reads (default is set as 10%);

(3) removing reads with overlap between the reads and the Adapter exceeding a certain threshold (default is set to be 15bp) and the mismatching number smaller than 3;

(4) for items such as small genome, if host contamination exists in the sample, the sample needs to be compared with a host database, and reads possibly derived from the host are filtered out.

And (3) performing genome assembly starting from Clean Data after quality control of each sample to obtain a sequence file capable of reflecting the basic situation of the sample genome, and evaluating an assembly result. The specific processing steps of genome assembly are as follows:

(1) obtaining clear Data after preprocessing, and assembling by using SOAP denovo assembly software:

selecting different K-mers (default selection 95, 107 and 119) for assembly, selecting the optimal kmer according to the type of the project,

selecting an assembly result of the minimum scaffold; screening again by utilizing the optimal kmer and adjusting other parameters (-d-u-R-F and the like) to obtain a primary assembly result;

(2) assembly was performed using SPAdes software:

selecting different K-mers (default selection 99 and 127) for assembly, selecting the optimal kmer according to the project type, and selecting the assembly result of the least scaffold;

(3) assembly was performed using abys software: selecting K-mer 64 for assembling to obtain an assembling result:

(4) integrating the assembly results of the three software by using CISA software, and selecting the assembly result of the least scaffold;

(5) hole filling is carried out on the preliminary assembly result by adopting gapplose software, and the same lane pollution is removed by filtering reads with low sequencing depth (less than 0.35 of average depth), so that the final assembly result is obtained;

(6) fragments below 500bp were filtered out and evaluated and statistically analyzed and subsequently gene predicted.

(7) The number and functional annotation of the functional Genes of the 22-2 strain was carried out by GO (Gene Ontology, http:// geneontology. org /), KEGG Kyoto Encyclopedia of Genes and Genomes, http:// www.genome.jp/KEGG /), COG (Cluster of organisms Groups of proteins, http:// www.ncbi.nlm.nih.gov/COG /), NR (NonRedundant Protein database), TCDB (Transporter Classification database).

Functional annotation the basic steps are as follows:

1) BLAST comparison is carried out on the prediction gene and each functional database (blastp, evalue is less than or equal to 1 e-5);

2) BLAST result filtering: for BLAST results of each sequence, the highest score alignment (default > 40%, coverage > 40%) was chosen for annotation. The annotation results are shown in FIGS. 1-6.

It is to be understood that the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and those skilled in the art should understand that they can make various changes, modifications, additions and substitutions within the spirit and scope of the present invention.

Claims (6)

1. A pesticide residue broad-spectrum degradation strain which is identified as bacillus cereus 22-2 (B)Bacillus cereus 22-2) and has been preserved in China center for type culture Collection with the preservation time of 26 months at 2021 and the preservation number of CCTCC NO: m2021936.

2. The Bacillus cereus 22-2 (B.cereus of claim 1)Bacillus cereus 22-2) application in degrading pesticide residue in soil or water body environment.

3. The use as claimed in claim 2, wherein the pesticides include imidacloprid, acetamiprid, triazolone, fosthiazate, chlorothalonil and cyhalothrin.

4. The Bacillus cereus 22-2 (B.cereus of claim 1)Bacillus cereus 22-2) application in producing pesticide residue degrading microbial inoculum.

5. Use of the Bacillus cereus 22-2 (B.cereus of claim 1)Bacillus cereus 22-2) produced degradation microbial inoculum.

6. The method for preparing the degradation microbial inoculum according to claim 5, which is characterized by comprising the following steps:

(1) inoculating the test tube liquid of bacillus cereus 22-2 cultured to logarithmic phase into a fermentation culture medium according to 0.5-1% of the volume of the fermentation culture medium, and culturing to logarithmic phase to obtain a fermentation strain;

(2) inoculating the prepared fermentation strain into a culture medium of a seed tank according to 5-10% of the volume of the culture medium of the seed tank, and culturing to logarithmic phase to prepare a seed solution;

(3) inoculating the seed liquid into the culture medium of a production tank according to 5-10% of the volume of the culture medium of a 5 ton production tank, culturing and fermenting, wherein the fermentation temperature is 32 ℃, the fermentation is stopped when the microscopic spore rate reaches more than 95%, and after the fermentation is finished, light calcium powder with the mass of 12.2% is added, and after centrifugation, spray drying is carried out to prepare a powder product with the bacterial load of 10 hundred million/g.

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