Agromyces strain, microbial inoculum and application of microbial inoculum

Abstract

The invention provides an Agromyces sp. Strain, an Agromyces sp. Fungicide and application thereof, and belongs to the technical field of microorganisms. The invention provides an Agromyces sp. Strain WXX04, and the preservation number of the Agromyces sp. Strain WXX04 is CGMCC (China General Microbiological Culture Collection Center) No. 36357. The Agromyces sp. Strain WXX04 is screened from rhizosphere soil of mulched peanuts for more than ten years in Shandong province, and can degrade a plurality of phthalic acid esters at the same time. The Agromyces sp. Strain WXX04 can effectively degrade phthalate, and can improve the remediation efficiency of phthalate pollution of the soil after being applied to the phthalate-polluted soil, the remediation efficiency of dibutyl phthalate and di (2-ethylhexyl) phthalate reaches 70% or above, and the remediation efficiency of the Agromyces sp. Strain WXX04 is up to 70% or above. The method has important significance for producing safe agricultural products by efficiently utilizing the phthalate-polluted cultivated land soil and realizing'simultaneous production and remediation '.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a strain of Agaricomyces, a microbial agent and application thereof. BACKGROUND

[0002] As a new pollutant with endocrine disrupting effect, phthalate acid esters (PAEs) are widely used in agricultural fields such as plastic film, packaging material, pesticide adjuvant, etc. PAEs are connected with plastic molecules by hydrogen bond or van der Waals force, and each other retains relatively independent chemical properties, so it is easy to age and decompose into soil during the use of various plastic products.

[0003] Microbial degradation is the mainstream technology for degrading PAEs in soil. Microorganisms can completely mineralize PAEs into harmless small molecules through specific enzyme systems, avoiding pollutant residues, and have the advantages of environmental friendliness, low cost, safety, etc. However, the current PAEs-degrading strains have generally weak environmental adaptability in the process of contaminated soil remediation. The degrading bacteria screened from non-cultivated soil media have insufficient colonization ability in PAEs-contaminated cultivated soil, and it is difficult to achieve efficient degradation of target pollutants. SUMMARY

[0004] The present application aims to provide a strain of Agaricomyces, a microbial agent and application thereof. The Agaricomyces strain and the microbial agent of the present application have strong adaptability and can effectively degrade phthalate acid esters.

[0005] The present application provides a strain of Agaricomyces (Agaricomyces sp.) WXX_04. Agromyces sp. The preservation number of the Agaricomyces strain WXX_04 is CGMCC No. 36357.

[0006] The present application also provides a microbial agent comprising the Agaricomyces strain WXX_04 described in the above scheme.

[0007] Preferably, the microbial agent comprises a bacterial suspension; the OD of the bacterial suspension is 0.8-1. 600 =0.8~1.

[0008] The present application also provides the application of the Agaricomyces strain WXX_04 described in the above scheme or the microbial agent in degrading phthalate acid esters, remediating phthalate acid ester-contaminated media and / or promoting crop growth.

[0009] Preferably, the medium is soil or water body.

[0010] Preferably, the concentration of phthalate acid esters in the phthalate acid ester-contaminated medium is 5-160 mg / L or 5-160 mg / kg.

[0011] Preferably, the phthalate comprises one or more of dimethyl phthalate, diethyl phthalate, diisobutyl phthalate, di(2-ethylhexyl) phthalate, dibutyl phthalate, and di-n-octyl phthalate.

[0012] Preferably, the phthalate comprises dibutyl phthalate and / or di(2-ethylhexyl) phthalate.

[0013] The present invention also provides a method for degrading phthalates, comprising the following steps: The *Pseudomonas* strain WXX_04 or the bacterial agent described in the above scheme is inoculated or applied to the medium containing phthalates to be degraded, and degradation is carried out.

[0014] Preferably, when the degradation medium is soil, the effective viable concentration of *Pseudomonas* strain WXX_04 inoculated in the soil is 10-1. 6 ~10 8 CFU / g.

[0015] This invention provides a strain of the genus *Pseudomonas* (…). Agromyces sp. The strain WXX_04, with the preservation number CGMCC No. 36357, was obtained from the rhizosphere soil of peanuts that had been covered with film for over ten years in Shandong Province. It can simultaneously degrade multiple phthalates. The WXX_04 strain is simple to cultivate, grows rapidly, is not prone to mutation, and has strong environmental adaptability. It effectively degrades phthalates, and when applied to phthalate-contaminated soil, it can improve the remediation efficiency of phthalate pollution in the soil. The remediation efficiency for dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) both reach over 70%, demonstrating excellent application potential. This invention is simple to operate, economical, easy to industrialize, and environmentally friendly. It is of great significance for efficiently utilizing phthalate-contaminated arable land to produce safe agricultural products and achieving "production and remediation simultaneously."

[0016] Biological Preservation Instructions New species of the genus *Pseudomonas* ( Agromyces sp WXX_04 was deposited on October 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36357. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a colony morphology diagram of strain WXX_04 on LB medium; Figure 2 A scanning electron microscope image of strain WXX_04; Figure 3 Phylogenetic tree of 16S rRNA gene of strain WXX_04; Figure 4 Phylogenetic tree of housekeeping genes for strain WXX_04; Figure 5 A statistical chart of COG annotations for strain WXX_04; Figure 6 GO annotation classification statistics for strain WXX_04; Figure 7 KEGG annotation classification statistics for strain WXX_04; Figure 8 This is a circumscribed genome diagram of strain WXX_04; Figure 9 The degradation effect of strain WXX_04 on DBP and DEHP in MSM; Figure 10 The degradation effect of strain WXX_04 on different PAEs in PAEs-contaminated soil; Figure 11 This shows the growth of peanuts at 30 days. Detailed Implementation

[0019] This invention provides a strain of the genus *Pseudomonas* (…). Agromyces sp. The strain WXX_04, whose preservation number is CGMCC No.36357, is described.

[0020] The soil mold strain WXX_04 of the application is a new species of soil mold, and is a phthalate ester degrading strain with strong adaptability and high phthalate ester degradation efficiency. The soil mold strain WXX_04 is obtained by directional screening of peanut rhizosphere soil of a PAEs polluted farmland through long-term mulching (mulching time ≥ 10 years), and is obtained through phthalate ester concentration gradient (5 mg / L→10 mg / L→20 mg / L→40 mg / L→80 mg / L→160 mg / L) domestication, and has high efficient degradation performance on phthalate ester: in an inorganic salt culture medium system, the degradation rate of the target substrate can be more than 90%; in a soil system, the phthalate ester degradation rate can be 52%~80%, and the degradation rates of dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) are the highest, and are 74% and 80%, respectively. The soil mold strain WXX_04 can solve the problem of phthalate ester plasticizer farmland pollution in long-term mulching agricultural production, provides a high efficient functional strain resource for bioremediation of phthalate ester pollution in farmland soil, and has significant practical value and broad application prospect.

[0021] In the application, the soil mold strain WXX_04 is a gram-positive bacterium, is cultured at 30 DEG C for 3 days on an LB culture medium, the colony is round, light yellow, wet, opaque, the surface is smooth, the edge is smooth and neat, and almost covers the plate after four days; under a scanning electron microscope, it is rod-shaped, and the length is 0.5~1 μm. The soil mold strain WXX_04 of the application can be cultured and grown in an inorganic salt liquid culture medium with dibutyl phthalate and di(2-ethylhexyl) phthalate as the only carbon source.

[0022] In the application, the nucleotide sequence of 16S rDNA of the soil mold strain WXX_04 is shown as SEQ ID NO. 1, and is specifically as follows: tttggagagt ttgatcctgg ctcaggacga acgctggcgg cgtgcttaac acatgcaagt 60 cgaacgatga acctggagct tgctctgggg gattagtggc gaacgggtga gtaacacgtg 120 agtaacctgc cctggactct gggataactt cgagaaatcg gagctaatac cggataggac 180 cttgcaccgc atggtgtggg gtggaaagtt tttcggtttg ggatggactc gcggcctatc 240 agcttgttgg tgaggtaatg gctcaccaag gcgtcgacgg gtagccggcc tgagagggtg 300 accggccaca ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat 360 attgcacaat gggcgcaagc ctgatgcagc aacgccgcgt gcgggatgac ggccttcggg 420 ttgtaaaccg cttttagtag ggaagaagcc ttcgggtgac ggtacctgca gaaaaaggac 480 cggctaacta cgtgccagca gccgcggtaa tacgtagggt ccgagcgttg tccggaatta 540 ttgggcgtaa agagctcgta ggcggtttgt cgcgtctgct gtgaaaacta gaggctcaac 600 ctctagcctg cagtgggtac gggcagactt gagtggtgta ggggagactg gaattcctgg 660 tgtagcggtg gaatgcgcag atatcaggag gaacaccgat ggcgaaggca ggtctctggg 720 cacttactga cgctgaggag cgaaagcgtg gggagcgaac aggattagat accctggtag 780 tccacgccgt aaacgttggg cgctagatgt ggggaccttt ccacggtttc cgtgtcgtag 840 ctaacgcatt aagcgccccg cctggggagt acggccgcaa ggctaaaact caaaggaatt 900 gacgggggcc cgcgcaagcg gcggagcatg cggattaatt cgatgcaacg cgaagaacct 960 TACCAAGGCT TGACATCACG AGAACCAGAA ATGGTCAACT CTTTGGACAC TC GTG 1020 AACAGGTGGT GCATGGTTGT CGTCAGCTCG TGTCTGTGAG ATGTGGGTTA GTCCCGCAA 1080 CGAGCGCAAC CCTCCTGCTA GTTTGCCAGC ACCTATGGGT GGGGACTCAT GTGAGACTGC 1140 CAGGGGTCAAC TCAGAGGAAG GTGGGGATGA CGTCAAAACA TCTGGCCCTT ATGTCTTGG 1200 GCTTCACGCA TGCTACAATG GCCGGTACAA AGGGCTGCGA TGTCTAAGGC GGAGCGAAT 1260 CCCAAAAAGC CGGTCTCAGT TCAGATTGAG GTCTGCAACT CGACCTCATG AAGTCGGAGT 1320 CGCTAGTAAT CGCAGATCAG CAACGCTGCG GTGAATACGT TCCCAGGCCT GTACACACC 1380 GCCCCTCAAG TCTATGAAAG TCAGTAACAC CTGAAGCCCG TGGCCTAACT CCTTGTGGAG 1440 AGCCGTCGAA GGTGGGATCG GTGATTAGGA CTAAGTCGTA CAAGGTAAGC CTTCCGGAA 1500 GTTGCTGGAT CACCTCCTTT 1523.

[0023] The application further provides a bacterial agent, comprising the strain WXX_04 of the genus Horghum as described in the above scheme.

[0024] As an embodiment, the bacterial agent comprises a bacterial suspension; the OD 600 of the bacterial suspension is 0.8-1.

[0025] The application further provides a preparation method of the bacterial agent as described in the above scheme, comprising the following steps: Inoculate the Agaric strain WXX_04 into LB liquid culture medium for expansion culture, centrifugal collection, and collection of the Agaric strain WXX_04 bacteria bodies; After washing the Agaric strain WXX_04 bacteria bodies 2-3 times with PBS buffer, resuspend in PBS to obtain the Agaric strain WXX_04 bacteria suspension, adjust the OD 600 to 0.8-1.0 to obtain the bacteria agent.

[0026] As an implementation form, the expansion culture temperature is 30℃; the expansion culture rotation speed is 180 r / min; the expansion culture time is 24 h; the centrifugal rotation speed is 8000 rpm; and the centrifugal time is 5 min.

[0027] The application further provides the use of the Agaric strain WXX_04 or the bacteria agent in the above scheme in the degradation of phthalate, the repair of phthalate contaminated medium, and / or the promotion of crop growth.

[0028] As an implementation form, the crop includes peanuts; and the promotion of crop growth includes the increase of peanut seedling biomass and / or plant height.

[0029] The Agaric strain WXX_04 can efficiently degrade phthalate during growth and has strong environmental adaptability, and can be used for bioremediation of phthalate pollution, especially for soil plasticizer pollution treatment in long-term mulching agricultural scenes, and ensures the safety of agricultural land. With people's attention to soil health, the demand for microbial organic fertilizer is gradually increasing. Therefore, the strain can be used as a microbial agent to produce a bacteria fertilizer integrating "repair-promotion" and has good application prospects in the field of plasticizer contaminated soil remediation.

[0030] As an implementation form, the medium is soil or water.

[0031] As an implementation form, the concentration of phthalate in the phthalate contaminated medium is 5-160 mg / L (aqueous solution) or 5-160 mg / kg (soil), further 10-80 mg / L or 10-80 mg / kg, and more further 20-40 mg / L or 20-40 mg / kg.

[0032] As an implementation form, the phthalate includes one or more of dimethyl phthalate, diethyl phthalate, diisobutyl phthalate, di(2-ethylhexyl) phthalate, dibutyl phthalate, and di-n-octyl phthalate.

[0033] The strain WXX_04 of the genus Paecilomyces can grow with the phthalate compounds as carbon source and energy source, and improve the accumulation of biomass, that is, the strain WXX_04 of the genus Paecilomyces can degrade the phthalate compounds described in the above scheme.

[0034] As an implementation form, the phthalate includes dibutyl phthalate and / or di(2-ethylhexyl) phthalate.

[0035] The application further provides a method for degrading phthalate, comprising the following steps: The strain WXX_04 of the genus Paecilomyces described in the above scheme or the microbial agent is inoculated or applied to a medium containing phthalate to be degraded, and degradation is performed.

[0036] As an implementation form, when the degradation medium is soil, the effective viable concentration of the strain WXX_04 of the genus Paecilomyces inoculated in the soil is 10 6 ~10 8 CFU / g.

[0037] In order to further illustrate the application, a strain of the genus Paecilomyces, a microbial agent and application thereof provided by the application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the protection scope of the application.

[0038] The medium formula in the embodiment of the application is as follows: The components of the inorganic salt liquid medium with dibutyl phthalate and di(2-ethylhexyl) phthalate as the only carbon source are as follows: 1.5 g / L of sodium dihydrogen phosphate dodecahydrate, 1.5 g / L of dipotassium hydrogen phosphate, 2.0 g / L of ammonium sulfate, 0.2 g / L of magnesium sulfate heptahydrate, 0.01 g / L of calcium chloride dihydrate, 0.001 g / L of ferrous sulfate heptahydrate, 1 mL / L of trace element solution, 100 mg / L of DBP and 100 mg / L of DEHP, and the pH value is 7.2; and 15 g / L of agar powder is added to the solid medium. The components of the trace element solution are as follows: 500 mg / L of disodium ethylenediaminetetraacetate, 10 mg / L of zinc sulfate heptahydrate, 5 mg / L of manganese sulfate, 30 mg / L of boric acid, 24 mg / L of cobalt sulfate heptahydrate, 5 mg / L of copper sulfate pentahydrate, 30 mg / L of sodium molybdate dihydrate, 50 mg / L of calcium hydroxide, and the pH value is 7.2.

[0039] The inoculation amount of the inorganic salt liquid medium degradation bacteria is 2% of the total system volume after inoculation, and the OD value of the bacterial suspension is OD 600 =0.8.

[0040] Luria-Bertani medium (LB): Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH 7.2; solid medium added agar 15 g / L.

[0041] Isolation and identification of strains in Example 1 (1) Isolation and screening of strains The rhizosphere soil samples of peanut with more than 10 years of long-term mulching in Shandong Province were collected, 200 g of which were stored in a brown glass bottle (250 mL) in the dark after being washed and dried, and were transported to the laboratory at 4°C. 10 g of the sample was weighed from the sampling site and added to 100 mL of MSM medium (PAEs concentration was 0 / 5 / 20 mg / L, the same below), which was placed in a shaking bed (30°C, 180 rpm) for 7 days. Then 2% of the enriched culture solution was inoculated into 100 mL of fresh MSM medium, and the gradient pressure acclimation method was used, and the transfer was performed every 7 days. The substrate PAEs was added gradually, i.e. the concentration gradient was 5, 10, 20, 40, 80, and 160 mg / L, and the operation was repeated.

[0042] After acclimation, a small amount of culture solution was taken with an inoculation needle and streaked onto solid MSM medium containing 5 / 20 mg / L PAES, and incubated in a constant temperature (30°C) incubator in the dark. The growth condition of the colonies on the plate was observed at regular intervals (every 24 h), and single colonies with good growth were selected and repeatedly streaked onto plates until they were purified. The purified single colonies were inoculated into 100 mL of LB liquid medium, and LB liquid medium without inoculation was used as a control. When the bacterial concentration reached OD 600 =0.8 (absorbance of the solution at 600 nm wavelength without centrifugation), 1 mL of bacterial suspension (resuspended in PBS buffer) was inoculated into 100 mL of MSM liquid medium containing 5 / 20 mg / L PAES, and the same conditions without inoculation were used as a control. After 7 days of incubation in a constant temperature shaking incubator (30°C, 180 rpm), the substrate residual amount was measured, and the degrading bacteria were selected. The above experiments were repeated four times.

[0043] Single colonies showing degradation effect were streaked again onto solid MSM agar plates (5 / 20 mg / L PAES), repeating the above steps until pure colonies with a uniform colony morphology were obtained. Following the purification standards in microbiological experiments, single colonies repeatedly streaked onto plates were picked up with an inoculation needle and transferred to a glass slide in a clean bench for microscopic examination to confirm their uniform bacterial shape. The purified strain was inoculated into LB liquid medium for overnight activation. 1.5 mL of the bacterial solution was centrifuged at 8000 rpm for 3 min, and then transferred to fresh, identical medium at a 1% inoculation rate (v / v). This enrichment and subculturing was performed four times. The degradation effect of the fifth-generation enrichment solution was assessed using GC-MS. The bacterial suspension effectively degrading PAEs was diluted and spread on LB solid medium containing 5 / 20 mg / L PAEs (DBP+DEHP), and incubated at 30°C for 5 days. Single colonies from the plates were picked and transferred to 10 mL of liquid LB test tube medium, then stored and transferred to 100 mL of MSM containing 5 / 20 mg / L PAEs (DBP+DEHP), and incubated at 30°C for 5 days. The final PAEs-degrading strain WXX_04 was obtained.

[0044] Morphological observation: WXX_04 was spread onto solid LB medium and incubated upside down at 30℃ for 3 days. Colonies were round, pale yellow, smooth, moist, opaque, and had neat edges. After four days, they almost completely covered the plate. See [link / description]. Figure 1 .

[0045] Electron microscopy sample preparation and observation: Step 1: Collect bacterial cells: Centrifuge the culture medium at 8000 rpm for 3-5 min, discard the supernatant, and add 2.5% glutaraldehyde fixative. The second step is fixation and washing: Fix with 2.5% glutaraldehyde for 4 h or overnight at 4°C. During this period, wash with 0.2 M PBS buffer (pH 6.8) 3 times, 15-20 min each time. The third step is dehydration and drying: treat with 30%, 50%, 70%, 85%, and 95% ethanol for 15-20 minutes in sequence, and finally treat with 100% ethanol for 10-15 minutes each time twice before freeze drying; Step 4: Conductivity treatment and measurement: First, firmly attach the sample to the conductive adhesive and ensure good grounding. Then, use an ion sputtering instrument or vapor deposition instrument to uniformly spray a layer of gold or platinum conductive film several nanometers to tens of nanometers thick on the sample surface. The purpose is to eliminate the sample charge effect, enhance the secondary electron emission signal, and obtain a high-quality image. After the treatment is completed, use SU8220 for instrument measurement.

[0046] like Figure 2 As shown in the scanning electron microscope, the WXX_04 cells are rod-shaped and about 1~2 μm long.

[0047] (2) Physiological and biochemical identification The physiological and biochemical characteristics of WXX_04 were identified, and the results are shown in Table 1.

[0048] Table 1. Identification of the physiological and biochemical characteristics of WXX_04

[0049] Note: "+" represents positive; "-" represents negative. (3) Molecular identification 16S rDNA molecular identification of strain WXX_04: Total bacterial DNA was extracted, and the bacterial genome was amplified by PCR using universal primers for bacterial 16S rDNA. After sequencing of the PCR products (performed by Shanghai Sangon Biotech), 20 strains most closely related at the species level were selected based on the 16S rRNA sequence, and a phylogenetic tree was constructed using the Neighbor-Joining (NJ) method with MEGA 6.0 software. It was found that WXX_04 is related to... Agromyces sp. Most similar ( Figure 3 A phylogenetic tree was constructed using the genomes of WXX_04 and its closest related type strain. The results showed that WXX_04 and... Agromyces sp. Gathered into a cluster ( Figure 4 Based on the morphological characteristics of WXX_04 and the comparative analysis of its 16S rRNA gene and genome phylogenetic tree, WXX_04 was ultimately identified as belonging to the genus *Pseudomonas*. Agromyces sp. ).

[0050] (4) Preservation Therefore, the inventor will use WXX_04 ( Agromyces sp. The strain WXX_04 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 27, 2025. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The accession number is CGMCC No. 36357.

[0051] Example 2: Genomic study of degrading strain WXX_04 WXX_04 cells were inoculated into liquid LB medium and cultured at 30°C and 180 rpm for 24 h on a shaker. The cells were then collected by centrifugation and sent to Shanghai Meiji Biotechnology Co., Ltd. on dry ice for genome sequencing to obtain the genomic information of WXX_04. The specific process is as follows: (1) DNA extraction WXX_04 strain was inoculated on LB solid medium plate and cultured at 30°C overnight. Single colony was picked up with inoculation needle and inoculated into 200 mL of LB liquid medium, cultured at 30°C in a shaker to logarithmic growth phase, the shaker speed was 180 r / min, then centrifuged at 12000 rpm, 4°C for 10 min, the supernatant was discarded and the bacterial cells were collected, and the culture medium in the bacterial cells was washed with sterile water. The genomic DNA was extracted according to the instructions of the bacterial DNA extraction kit (magnetic bead method) (T07-100, China). The purified genomic DNA was quantified, and the high-quality DNA was used for the following library sequencing.

[0052] (2) Whole genome sequencing The strain WXX_04 was sequenced using PacBio Sequel IIe and illumina sequencer (NovaSeq6000). For Illumina library construction, genomic DNA was fragmented into 400 bp fragments, and the fragment size distribution was identified by agarose gel. The NEXTFLEX Rapid DNA-Seq Kit was used for library preparation. The data generated by PacBio Sequel IIe and Illumina sequencing platform were subjected to bioinformatics analysis. For Illumina sequencing, the prepared library was subjected to double-end sequencing (2x150 bp) on the illumina sequencer (NovaSeq6000). For PacBio sequencing, the single-stranded loop of the library was annealed and combined with the polymerase at the bottom of the PacBio Sequel IIe sequencing instrument fixed ZMW (zero-mode waveguides, zero-mode waveguide hole), and the sequencing reaction reagent was added. After each base pairing synthesis, the corresponding light will be emitted and detected, and each base synthesis will be displayed as a pulse peak. Coupled with a high-resolution optical detection system, real-time detection was performed. All analyses were performed on the Shanghai Majorbio Cloud Platform (http: / / cloud.majorbio.com).

[0053] The whole genome length of WXX_04 is 3,819,870 bp, including one chromosome. The G+C content of the chromosome is 72.16%. The total length of the gene base is 3,512,556 bp, and 3,580 predicted protein coding sequences (CDS) are identified. The average length of the coding genes is 981.16 bp, and all genes are estimated to cover 91.95% of the whole genome. The chromosome also encodes 47 tRNAs and 6 rRNAs including 5S rRNA, 16S rRNA and 23S rRNA 3 types. The specific information is shown in Table 2.

[0054] Table 2. Coding gene prediction statistics table

[0055] (3) Whole genome annotation The Illumina data and HiFireads after quality control were assembled by Unicycler v0.4.8 (Wick R R, Judd LM, Gorrie C L, et al. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads [J]. PLoS computational biology, 2017, 13(6), e1005595.) in this sample. The coding sequences (CDS) in the genome were predicted by Glimmer or Prodigal v2.6.3 (Hyatt D, Chen G L, LoCascio P F, et al. Prodigal: prokaryotic gene recognition and translation initiation site identification [J]. BMC bioinformatics, 2010, 11(1), 1-11), and the plasmid genes were predicted by GeneMarkS (Besemer J, Borodovsky M. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses [J]. Nucleic Acids Research, 2005, 33(Web Server):W451-W454.) software, tRNAscan-SE v2.0 (Chan, P.P. and Lowe, T.M. (2019) tRNAscan-SE: Searching for tRNA Genes in Genomic Sequences. Methods Mol Biol. 1962:1-14.) for tRNA prediction, and Barrnap v0.9 (https: / / github.com / tseemann / barrnap) for rRNA prediction. The predicted CDS were functionally annotated from GO, COG, KEGG databases by using BLASTP, Diamond, HMMER and other sequence alignment tools, and the secondary metabolite synthesis gene clusters were predicted by antiSMASH v5.1.2.

[0056] 1) COG annotation Strain WXX_04 has 2919 functional genes assigned to the COG database, accounting for 81.54% of the total genes. For example... Figure 5 It is evident that a significant number of proteins are involved in amino acid transport and metabolism (E) and transcription (K), accounting for 13.77% and 12.16% of the predicted functional genes, respectively. This is followed by carbohydrate transport and metabolism (G), accounting for 10.17%. 276 proteins were classified as general function predictions (R), accounting for 9.46%. Relatively fewer proteins are involved in inorganic ion transport and metabolism (P); coenzyme transport and metabolism (H); translation; and ribosomal structure and biogenesis (J), accounting for 7.71%, 7.16%, and 6.89%, respectively.

[0057] 2) GO comments After summarizing and statistically analyzing the functions of the CIP genes in strain WXX_04 from three aspects—cellular component, biological process, and molecular function—a total of 562 GO annotations were obtained. Of these, 49 annotations were related to cellular components (8.72% of the total), 232 were related to biological processes (41.28%), and the remaining 281 annotations were related to molecular function (50%). The GO annotation information was simplified to obtain GOslim classifications. The top 20 most annotated GOslim subclasses within each subclass were plotted, as shown below. Figure 6 As shown.

[0058] The number of genes related to membrane is the largest, 84; the number of genes related to plasma membrane is also large, reaching 73; the number of genes related to cytosol is 60; the number of genes related to cytoplasm is 37; the remaining annotated genes are mainly related to cytoplasmic ribosomal large subunit, ATP-binding component (ABC) transporter complex, protein DNA complex extracellular membrane, cell division site, etc. In the biological process gene annotation, the largest number is translation, 22; followed by transmembrane transport, 19; the number of carbohydrate metabolic process annotation genes is 11; in addition, the biological process also includes the regulation of DNA template transcription, negative regulation of DNA template transcription, proteolysis, response to amino acids, response to stress, etc. In the molecular function annotation, the number of DNA binding genes is the largest, 47; followed by ATP binding annotation genes, 44; the number of DNA-binding transcription factor activity annotation genes is 40; the remaining annotated genes mainly include metal ion binding, ATP decomposition activity, ribosomal structural composition, transmembrane transporter activity, etc.

[0059] 3) KEGG annotation KEGG database annotated 2629 functional genes in the genome of strain WXX_04. Through Figure 7 It can be seen that the annotation results are divided into 6 categories, including Cellular Processes, Environmental Information Processing, Genetic Information Processing, Human Diseases, Metabolism and Organismal Systems. Among them, the number of genes related to metabolism is the largest, reaching 2236, accounting for 85.05%, followed by environmental information processing functions (such as signal transduction, membrane transport, etc.) and cellular processes (such as cell colony-prokaryotes, cell movement, transport and catabolism, etc.), the number of related genes is 271. In the metabolic functional genes, global and overview map (875), amino acid metabolism (284) and carbohydrate metabolism (274) are the three main secondary classifications.

[0060] (4) Bacterial genome circle map The genome circle map can comprehensively show the characteristics of the genome, such as the distribution of genes on the positive and negative strands, the COG functional classification of the genes, the GC content, the genomic island, the homologous genes, etc. Various information (here referring to the GC content, the GC bias, the tRNA / rRNA, the COG annotation, the base modification and the restriction modification system related enzyme) is integrated and drawn in a genome circle map, and the drawing software used is Circos.

[0061] Figure 8 The genome circle map of the strain WXX_04 is shown, the outermost circle of the circle map is the identification of the genome size; the second and third circles are the CDS on the positive and negative strands, and different colors represent different COG functional classifications of the CDS; the fourth circle is the rRNA and tRNA; the fifth circle is the GC content; the outward red part represents that the GC content of the region is higher than the average GC content of the whole genome, and the higher the peak value represents the greater the difference from the average GC content, and the inward blue part represents that the GC content of the region is lower than the average GC content of the whole genome, and the higher the peak value represents the greater the difference from the average GC content; the innermost circle is the GC-Skew value, the specific algorithm is G-C / G+C, which can assist in judging the leading strand and the lagging strand, generally the GC skew of the leading strand > 0, and the GC skew of the lagging strand < 0, and can also assist in judging the replication origin (cumulative offset minimum value) and the termination point (cumulative offset maximum value), which is most important for circular genomes.

[0062] The total length of the genome of the strain WXX_04 is 3,819,870 bp, the GC content is 72.16%, and it contains 3580 CDS. 2919 CDS are annotated by COG; 503 CDS are annotated by GO; 2629 CDS are annotated by KEGG.

[0063] In addition, the strain WXX_04 is also involved in the metabolic degradation pathways of various different types of compounds, a total of 110 genes are involved in 16 exogenous compound metabolic pathways, and there are rich exogenous substance degradation genes, and the specific degradation information is shown in Table 3, so it can be speculated that there are a large number of unknown function genes in the strain WXX_04 which can be further analyzed and mined.

[0064] Table 3: Gene prediction statistics table

[0065] Example 3: WXX_04 degradation effect experiment of phthalate esters in inorganic salt (MSM) (1) Preparation of bacterial suspension: the purified WXX_04 was inoculated into 10 mL of LB liquid medium and activated overnight to the logarithmic phase, then centrifuged at 8000 rpm for 5 min to collect the bacterial cells, washed twice with PBS, resuspended, and adjusted to OD 600 = 0.8~1 as the bacterial suspension.

[0066] (2) Determination of degradation performance of the strain: 2 mL of the strain was inoculated into 100 mL of MSM medium containing 100 mg / L of phthalate (100 mg / L of dibutyl phthalate and 100 mg / L of di(2-ethylhexyl) phthalate), and no inoculation was used as a control, with four replicates in each group. The culture was incubated at 30℃ in a constant temperature shaker at 180 rpm for 7 days, and the substrate concentration and strain growth (OD 600 ) were determined by GC / MS every day, and the determination was performed until 120h.

[0067] (3) GC-MS detection of dibutyl phthalate and di(2-ethylhexyl) phthalate: 6 mL of the sample to be tested was taken, 3 mL of n-hexane + acetone (9:1) was added for extraction twice, the organic phase was combined, anhydrous Na2SO4 was used for dehydration, and the filtered paper was concentrated to 0.5~1 mL by nitrogen blowing, and then detected by GC-MS. The conditions of the chromatography are as follows: the inlet temperature is 280℃, the injection mode is splitless injection, the carrier gas is helium, the injection amount is 1 μL, and the temperature program is as follows: the initial column temperature is 50℃, maintained for 1 min, then increased to 200℃ at a rate of 15℃ / min, maintained for 1 min, then increased to 280℃ at a rate of 8℃ / min, maintained for 3 min. The mass spectrometry conditions are as follows: the ionization mode is electron impact source (EI), the ion source temperature is 230℃, the ionization energy is 70 eV, the mass spectrum interface temperature is 280℃, the quadrupole rod temperature is 150℃, the mass scan range is 35U~450U, and the data acquisition mode is SIM.

[0068] The samples taken at regular intervals were identified by GC-MS, and the degradation results of dibutyl phthalate and di(2-ethylhexyl) phthalate are shown in Figure 9 by the 7th day, the degradation rate of DBP in the MSM medium reached 98.11%, and the degradation rate of DEHP reached 97.44%.

[0069] Example 4: Degradation effect of WXX_04 on phthalate in soil (1) Soil sample treatment: The PAEs naturally contaminated soil sample was sieved (screen mesh size 2 mm) to remove large particle impurities and ensure uniform soil. The humidity of the soil was adjusted to 50%~60% to ensure that the soil was not dry. The soil was divided and placed in glass culture bottles, 200 g per culture bottle.

[0070] (2) Preparation of bacterial suspension: the purified WXX_04 was inoculated into 150 mL of LB liquid medium and activated overnight to the logarithmic phase, the bacterial cells were collected by centrifugation at 8000 rpm for 5 min, washed with PBS twice, resuspended, and adjusted to OD 600 = 0.8~1 as the bacterial suspension.

[0071] (3) Bacterial inoculation: 10 6 ~10 8 CFU / g of soil was inoculated, and the non-inoculated soil was used as a control, with four replicates in each group. After 15 d of culture, the phthalate concentration was determined by GC / MS.

[0072] (4) GC-MS detection of phthalates: 5 g of soil sample was added to 30 mL of n-hexane + acetone (1:1) mixed solvent, vortexed, and then placed for 12 h at 25℃, 100 kHz ultrasonic extraction for 30 min, 3000 r / min centrifugation for 5 min, filtered into a flask, and then 15 mL of n-hexane + acetone (1:1) mixed solvent was added and ultrasonicated for 15 min, repeated twice, the supernatant was combined in a flask, concentrated to about 1 mL, mixed with n-hexane, and concentrated to about 1 mL under the same conditions, and then identified by GC-MS.

[0073] The conditions of the chromatography were as follows: the injection port temperature was 280℃, the injection mode was splitless injection, the carrier gas was helium, the injection volume was 1 uL, and the temperature program was as follows: the initial column temperature was 50℃, maintained for 1 min, then increased at a rate of 15℃ / min to 200℃, maintained for 1 min, and then increased at a rate of 8℃ / min to 280℃, maintained for 3 min. The mass spectrometry conditions were as follows: the ionization mode was electron impact source (EI), the ion source temperature was 230℃, the ionization energy was 70 eV, the mass spectrometry interface temperature was 280℃, the quadrupole rod temperature was 150℃, the mass scan range was 35U-450U, and the data acquisition mode was SIM.

[0074] The phthalate content in the soil samples at 15 d was determined by GC-MS, and the degradation rate is shown in Table 4. Figure 10 Compared with the non-inoculated soil samples, the bacterial inoculation treatment can significantly reduce the content of phthalates, and the degradation rate is significantly improved compared with the control. Specifically, the degradation rate of dibutyl phthalate is increased by 31 percentage points (from 43% to 74%), the degradation rate of di(2-ethylhexyl) phthalate is increased by 33 percentage points (from 47% to 80%), the degradation rate of dimethyl phthalate is increased by 19 percentage points (from 39% to 58%), and the degradation rate of diisobutyl phthalate is increased by 15 percentage points (from 37% to 52%).

[0075] Table 4 Residual amount of phthalate esters in soil (mg / kg)

[0076] Example 5 Test of the degradation effect of WXX_04 on peanut growth and phthalate esters in peanuts (1) Soil sample treatment: The PAEs contaminated soil sample was sieved (screen mesh size 2 mm) to remove large particle impurities and ensure uniformity of the soil. The soil was divided and placed in clay pots (pot bottom 12 cm, pot mouth 18 cm, height 17 cm), with 1.5 kg (dry soil) of soil per pot. The soil moisture was adjusted to between 50% and 60% to ensure that the soil was not dry.

[0077] (2) Preparation of bacterial suspension: The purified WXX_04 was inoculated into 10 mL of LB liquid medium and incubated overnight to the logarithmic phase. The bacterial cells were collected by centrifugation at 8000 rpm for 5 min, washed twice with PBS, and resuspended to adjust the OD 600 = 0.8 as the bacterial suspension.

[0078] (3) Bacterial inoculation: WXX_04 bacterial agent was applied to the clay pots at 10 6 ~10 8 CFU / g of soil, and peanut seeds were planted in the pots. There were four replicates in each group. After 30 days of culture, the growth of the peanuts was observed, the samples were cleaned, freeze-dried, ground, and passed through a 60-mesh sieve. The phthalate ester concentration was determined by GC-MS after treatment.

[0079] (4) Detection of phthalate esters in plants by gas chromatography-mass spectrometry (GC-MS): 5 g of plant sample was added to 30 mL of dichloromethane, vortexed, and allowed to stand for 12 h at a water temperature of 25℃. The sample was ultrasonically extracted for 30 min at 100 kHz, centrifuged at 3000 r / min for 5 min, filtered into a flask, and washed with 10 mL of dichloromethane in several portions. The combined filtrate was concentrated to about 1 mL and identified by GC-MS.

[0080] The conditions of the chromatography were as follows: the injection port temperature was 280℃, the injection mode was splitless injection, the carrier gas was helium, the injection volume was 1 μL, and the temperature program was as follows: the initial column temperature was 50℃, maintained for 1 min, then increased at a rate of 15℃ / min to 200℃, maintained for 1 min, and then increased at a rate of 8℃ / min to 280℃, maintained for 3 min. The mass spectrometry conditions were as follows: the ionization mode was electron impact source (EI), the ion source temperature was 230℃, the ionization energy was 70 eV, the mass spectrometry interface temperature was 280℃, the quadrupole rod temperature was 150℃, the mass scan range was 35U~450U, and the data acquisition mode was SIM.

[0081] The growth of peanuts at 30 d is shown in Table 5. Figure 11 As shown in Table 5, the plant height and biomass data of peanuts are shown in Table 5. Compared with the non-inoculated peanut samples, the inoculation treatment can significantly promote the growth of peanuts, increase the plant height and increase the biomass. The plant height of peanuts is increased by 24.19%, and the aboveground and underground biomass is increased by 8.15% and 16.82%, respectively.

[0082] The phthalate esters in peanut samples at 30 d were determined by GC-MS, and the phthalate ester content in the underground and aboveground parts of peanuts is shown in Table 6. Compared with the non-inoculated peanut samples, the inoculation treatment can significantly reduce the phthalate ester content in peanuts, and the content of various phthalate esters in peanuts is significantly reduced compared with the control. Specifically, compared with the control, the dibutyl phthalate content in the underground part of peanuts treated with WXX_04 is reduced by 21.28%, the di(2-ethylhexyl) phthalate is reduced by 44.04%, the dimethyl phthalate is reduced by 57.09%, the diisobutyl phthalate is reduced by 36.04%, and the di-n-octyl phthalate is reduced by 37.19%; the dibutyl phthalate content in the aboveground part of peanuts is reduced by 32.55%, the di(2-ethylhexyl) phthalate is reduced by 26.01%, the dimethyl phthalate is reduced by 34.66%, the diisobutyl phthalate is not detected, and the di-n-octyl phthalate is reduced by 20.87%.

[0083] Table 5 Plant height and biomass of peanuts

[0084] Table 6 Phthalate ester content in peanuts (mg / kg)

[0085] Although the above embodiments make a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which are within the protection scope of the present application.

Claims

1. A strain of the genus Paenibacillus (Paeinobacillus sp.) strain WXX_04, characterized in that, Agromyces sp. The preservation number of the Agromyces sp. strain WXX_04 is CGMCC No. 36357. ​ 2. An inoculant characterized in that, The Agromyces sp. strain WXX_04 of claim 1.

3. The bacterial agent of claim 2, wherein The bacterial agent comprises a bacterial suspension; the OD 600 = 0.8~1.

4. Use of the Agromyces sp. strain WXX_04 of claim 1 or the microbial inoculum of claim 2 or 3 in degrading phthalate, remediating phthalate-contaminated medium and / or promoting crop growth.

5. Use according to claim 4, characterized in that, The medium is soil or water.

6. Use according to claim 4, characterized in that, The concentration of phthalate in the phthalate-contaminated medium is 5-160 mg / L or 5-160 mg / kg.

7. Use according to claim 4, characterized in that, The phthalate includes one or more of dimethyl phthalate, diethyl phthalate, diisobutyl phthalate, bis(2-ethylhexyl) phthalate, dibutyl phthalate and di-n-octyl phthalate.

8. Use according to claim 7, characterized in that, The phthalate includes dibutyl phthalate and / or bis(2-ethylhexyl) phthalate.

9. A method of degrading phthalates, characterized by, The method comprises the following steps: The Agromyces sp. strain WXX_04 of claim 1 or the microbial inoculum of claim 2 or 3 is inoculated or applied to the phthalate-containing medium to be degraded for degradation.

10. The method of claim 9, wherein, When the degradation medium is soil, the effective viable cell concentration of the strain of the genus Paecilomyces inoculated in the soil is 10 6 ~10 8 CFU / g.

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