Matuota novosphingobium hpd1 for degrading high-efficiency fluazifop-butyl acid and application thereof

Abstract

The present application relates to the field of microbial technology, and particularly relates to a strain of Matuura sphingolipid bacteria HPD1 for degrading high-efficiency fluazifop-butyl acid and application thereof, the Matuura sphingolipid bacteria HPD1 is classified and named as Novosphingobium mathurense , which was preserved in the China General Microbiological Culture Collection Center on May 9, 2026, with a preservation number of CGMCC No. 38610 and a preservation address of No. 1, Beichen West Road, Chaoyang District, Beijing. The Matuura sphingolipid bacteria HPD1 and the microbial inoculant thereof can effectively degrade high-efficiency fluazifop-butyl acid in a wide pH and temperature range, can be used as excellent functional bacteria for degrading pesticide residues, and can be used in the biological remediation of residual high-efficiency fluazifop-butyl acid in water bodies, contaminated soil, coastal wetland sediments and Spartina alterniflora treatment areas, and can provide functional bacteria resources that can be repeatedly obtained and environmentally friendly microbial remediation technology.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Matura sphingosine monocytogenes HPD1 for degrading highly efficient flupyradifurone and its applications. Background Technology

[0002] In recent years, chemical control has become a popular method for controlling the invasive plant Spartina alterniflora (Spartina alterniflora) in coastal salt marshes and wetlands. Spartina alterniflora This is an important means of control. Among them, haloxyfop-P-methyl (HPM, trade name "High-Efficiency Garlic") is one of the commonly used selective herbicides for controlling Spartina alterniflora in estuaries and coastal wetlands. This herbicide belongs to the aryloxyphenoxypropionate class of herbicides and is a post-emergence foliar selective herbicide. Its molecule contains a methyl ester structure, which is highly lipid-soluble, facilitating penetration of the cuticle of plant leaves and absorption by plant tissues. After entering plant tissues, its methyl ester group is hydrolyzed into the corresponding free acid form—haloxyfop-P acid (HP), which mainly exerts acetyl-CoA carboxylase (ACCase) inhibitory activity, blocking fatty acid synthesis and meristematic growth in plants. Due to its high selectivity for gramineous plants, HPM has shown good control effects against Spartina alterniflora and is currently widely used in the large-scale control of invasive Spartina alterniflora in estuaries and coastal wetlands in China.

[0003] It is important to note that HPM parent material is usually not the primary form of long-term environmental residues. After entering plant debris, soil, sediment, and tidal flats, HPM undergoes chemical hydrolysis and is further converted to HP by microbial esterases. This conversion process is essentially the breaking of methyl ester bonds, generating acidic products with free carboxylic acid structures. Compared to HPM, HP has higher water solubility and lower volatility. In neutral to weakly alkaline wetland pore water environments, HP is more likely to exist in ionized form, thus possessing the potential to migrate into pore water, sediment-water interfaces, and tidal exchange waters. Therefore, a rapid decrease in HPM concentration in environmental samples does not necessarily mean the simultaneous elimination of its pollution risk; its conversion product, HP, as the active ingredient, better reflects the actual exposure level of this herbicide in wetland ecosystems.

[0004] With the completion of the phased tasks of the "Special Action Plan for the Control of Spartina alterniflora (2022-2025)," my country's Spartina alterniflora management has shifted from a phase of concentrated eradication to a new phase emphasizing "dynamic zeroing, long-term management, and ecological restoration." Relevant national departments have proposed achieving "dynamic zeroing" of Spartina alterniflora nationwide by 2030, while continuously promoting monitoring and early warning, long-term management, and zoned and categorized ecological restoration. Against this backdrop, the main residual form of HP, rapidly transformed from HPM after chemical control, may pose a risk of migration and exposure during sediment pore water, sediment-water interfaces, and tidal water exchange in coastal wetlands. Therefore, developing green remediation technologies that can promote HP degradation in coastal wetland environments is of great significance for reducing the residual risks after chemical control.

[0005] Microbial remediation technology has advantages such as minimal environmental disturbance, relatively low cost, strong targeting, and low risk of secondary pollution, making it an important technological direction for the ecological remediation of pesticide residues. Currently, research on the microbial degradation of HPM mainly focuses on the "methyl ester hydrolysis" stage, that is, the conversion of HPM into HP by carboxylesterases produced by bacteria or fungi. Existing reports... Aquamicrobium , Corynebacterium , Brevundimonas Strains and their associated esterases can catalyze the ester bond cleavage of various aryloxyphenoxypropionate herbicides, demonstrating good conversion capabilities for HPM and other methyl ester precursors. However, this process essentially converts the applied form into an environmentally active acid form, rather than achieving further degradation of HP free acids. In contrast, microbial resources capable of directly utilizing or efficiently converting HP free acids remain relatively limited. Therefore, screening functional microorganisms adapted to wetland sediment environments and capable of directly degrading HP, constructing highly efficient microbial agents, and elucidating their degradation products and transformation pathways are of significant scientific importance and application prospects for reducing the residual risk of major transformation products after HPM application and improving the safety and long-term stability of coastal wetland ecological restoration after chemical control. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a strain of *Sphingosine Matura* HPD1 that has low environmental disturbance, relatively low cost, strong targeting, low risk of secondary pollution, adaptability to wetland sediment environment, stable and efficient degradation ability, and rapid degradation, for the degradation of highly efficient flupyradifurone, and its application.

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is: *Sphingosine Maturae* for degrading highly efficient flupyradifurone (SMR). Novosphingobium mathurense HPD1, the *Sphingosine Maturae* HPD1, was deposited on May 9, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.38610, located at No. 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The present invention also provides a microbial agent for degrading highly efficient flupyradifurone, the microbial agent comprising the above-mentioned Sphingosine Matura sphingolipidii HPD1 for degrading highly efficient flupyradifurone.

[0009] As a preferred technical solution, the microbial agent can rapidly degrade highly efficient flupyradifurone, with a total degradation rate of over 99%.

[0010] As a preferred technical solution, the pH of the environment in which the microbial agent degrades highly efficient flupyradifurone is 5.0 to 9.0.

[0011] As a preferred technical solution, the suitable temperature for the microbial agent to degrade highly efficient flupyradifurone is 25-30°C.

[0012] As a preferred technical solution, the degradation rate of highly efficient flupyradifurone by the microbial agent increases with the increase of the inoculum amount.

[0013] As a preferred technical solution, the microbial agent can maintain stable degradation activity in the complex sediment micro-ecological environment.

[0014] This invention also provides a method for preparing the above-mentioned microbial agent for degrading highly efficient flupyradifurone. The method involves inoculating *Sphingosine Maturae* HPD1 into 100 mL of NB liquid medium and incubating it at 30°C and 120 rpm with shaking for 12–16 h to obtain a bacterial suspension. The bacterial suspension is then aliquoted into 50 mL sterile centrifuge tubes and centrifuged at 4°C and 4000 rpm for 30 min to collect the bacterial cells. The bacterial cells are washed three times with PBS buffer to remove residual culture medium components, and then resuspended in basal salt liquid medium, with OD adjusted. 600 The standard seed solution, i.e., the microbial inoculant, is prepared by reaching version 1.0.

[0015] Due to the adoption of the above technical solution, the beneficial effects of this invention are as follows: The *Neosphophytum matura* HPD1 and its microbial agent provided by this invention can effectively degrade highly effective flupyradifurone (FOD) within a wide pH and temperature range. They can serve as excellent functional bacteria for degrading pesticide-related residues and can be used for the bioremediation of FOD residues in water bodies, polluted soils, coastal wetland sediments, and *Spartina alterniflora* control areas. This invention provides a reproducible functional bacterial resource and an environmentally friendly microbial remediation technology for the treatment of FOD residue pollution. Attached Figure Description

[0016] The following figures are intended only to illustrate and explain the present invention and do not limit the scope of the invention. Wherein: Figure 1 This is the LC-MS / MS quantitative standard curve of HP; Figure 2 This is a colony morphology diagram of *Matula neosphingosine monocytogenes* HPD1, as described in this invention. Figure 3 This invention relates to a phylogenetic tree of *Neosphingosine Matura* HPD1 constructed based on the 16S rRNA sequence; Figure 4 This invention is a phylogenetic tree of *Neosphomonas matulata* HPD1 constructed based on the whole genome sequence; Figure 5 This is a graph showing the degradation effect of the microbial agent of the present invention on HP over time; Figure 6 This is a graph showing the degradation efficiency of HP by the microbial agent of the present invention under different pH conditions; Figure 7 This is a graph showing the degradation efficiency of HP by the microbial agent of the present invention under different temperature conditions; Figure 8 This is a graph showing the effect of different inoculum amounts of microbial agents on HP degradation efficiency. Figure 9 This is a diagram showing the degradation effect of the microbial agent of the present invention on HP in the surface sediments of coastal wetlands; Figure 10 This is a schematic diagram of the main transformation products of HP degradation by the microbial agent of this invention and its predicted degradation pathway; Figure 11 This is a circled genome diagram of the *Neosphingosine Matura* HPD1 strain of the present invention; Figure 12 This is a COG classification statistical diagram of the *Neosphomonas matulata* HPD1 strain of the present invention; Figure 13 This is a statistical graph of GO functional annotations for the *Matula neosphingosine* HPD1 strain of the present invention; Figure 14 This is a statistical chart of KEGG functional annotations for the *Matura neosphingosine* HPD1 strain of the present invention. Detailed Implementation

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments. In the following detailed description, only certain exemplary embodiments of the invention are described by way of illustration. Undoubtedly, those skilled in the art will recognize that various modifications can be made to the described embodiments without departing from the spirit and scope of the invention. Therefore, the drawings and description are illustrative in nature and not intended to limit the scope of the claims.

[0018] To make the objectives and technical solutions of this invention clearer and easier to understand, the invention will be further described in detail below.

[0019] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0020] Unless otherwise specified in the following embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual shall apply.

[0021] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0022] Example 1: Enrichment of highly efficient fluopyram-degrading bacteria To enrich microorganisms capable of degrading haloxyfop-P acid (HP), this example uses the root system of reeds from coastal salt marshes as the microbial source. Healthy reed plants were selected, and their roots were carefully separated from the soil. Using sterile scissors, 5–10 cm root segments were cut at the root-stem junction of the reed plants and transferred to sterile centrifuge tubes pre-filled with 30 mL of autoclaved phosphate-buffered saline (PBS). The centrifuge tubes were then sonicated for 30 s to fully release the rhizosphere-attached microorganisms, obtaining a PBS suspension containing rhizosphere microorganisms. 500 μL of this suspension was inoculated into 50 mL of minimal salt medium (MSM) containing 50 mg / L HP. The MSM medium formula is as follows: disodium hydrogen phosphate 2800 mg / L, potassium dihydrogen phosphate 1000 mg / L, ammonium sulfate 500 mg / L, magnesium chloride 53 mg / L, calcium salt tetrahydrate 50 mg / L, disodium EDTA 0.5 mg / L, ferrous sulfate heptahydrate 0.2 mg / L, zinc sulfate heptahydrate 0.01 mg / L, manganese chloride tetrahydrate 0.003 mg / L, boric acid 0.03 mg / L, cobalt chloride hexahydrate 0.02 mg / L, copper chloride dihydrate 0.001 mg / L, nickel chloride hexahydrate 0.002 mg / L, sodium molybdate dihydrate 0.003 mg / L.

[0023] After inoculation, the liquid culture medium was cultured at 30°C and 120 rpm for 5 days in the dark with shaking (the dark treatment was to prevent photodegradation of *HP*), yielding the bacterial suspension. To enrich the degrading bacterial community using *HP* as the sole carbon source, a continuous subculturing strategy was adopted: after each culture, the inoculum was transferred at a rate of 1% (v / v) to fresh MSM medium containing 50 mg / L *HP*, and cultured under the same conditions. Each subculturing cycle was 5 days, and a total of 9 subculturings were performed to gradually enrich a microbial community with stable *HP* degradation capabilities.

[0024] During the enrichment process, the residual concentration of HP in the bacterial culture was periodically detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0025] Specifically, 500 μL of the enriched bacterial culture was taken, 400 μL of formic acid was added and mixed thoroughly, followed by 5 mL of acetonitrile. After vortexing for 3 min, 0.2 g of NaCl was added and vortexed for 3 min. The mixture was then centrifuged at 6000 r / min for 5 min, and the supernatant was collected and filtered through a 0.22 μm organic phase nylon filter membrane. The filtrate was used for LC-MS / MS analysis. The chromatographic conditions were as follows: an Athena UHPLC C18 column (LAEQ-2105UA, 3 μm, 2.1 × 50 mm) was used; the column temperature was set at 40°C; the injection volume was 10 μL; and the mobile phase and elution conditions are detailed in Table 1.

[0026]

[0027] The mass spectrometry parameters were set as follows: positive ion mode was used; ion source temperature (TEM) was 500°C; ion spray voltage (IS) was 4500 V; curtain gas (CUR) flow rate was 10 mL / min; nebulizer gas (GS1) flow rate was 60 mL / min; auxiliary gas (GAS2) flow rate was 60 mL / min; collision gas (CAD) flow rate was 8 mL / min; collision chamber outlet voltage (CXP) was 10 eV; the specific parameters for detecting ion pairs, declustering voltage (DP), and collision energy (CE) are shown in Table 2.

[0028]

[0029] A series of standard solutions with mass concentrations of 0.5, 1, 5, 10, 20, 50, 100, and 200 ng / mL were prepared by serially diluting a 100 mg / L HP standard solution with acetonitrile. The standard solutions at each concentration were analyzed using the LC-MS / MS method described above, and the corresponding peak areas were recorded (see Table 3). A standard curve was plotted with concentration on the x-axis and peak area on the y-axis (see Table 3). Figure 1 This standard curve is used for the quantitative analysis of residual *HP* in bacterial culture by periodic determination of the amount in the culture solution during the enrichment process using liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0030]

[0031] Example 2: Screening and identification of highly efficient fluopyram-degrading bacteria - *Neosphomonas matulata* HPD1 1. Strains screening Take 500 μL of the bacterial suspension obtained from the ninth generation enrichment culture in Example 1, and spread it evenly on an MSM solid medium plate containing 50 mg / L HP, with three replicates. Incubate the plates in a 30°C incubator in the dark. After colonies have grown, select single colonies that can grow stably on the HP-containing MSM solid medium and streak them five times on the same medium until a morphologically consistent pure culture is obtained, named HPD1 strain. This strain can grow in a medium with HP as the sole carbon source, preliminarily indicating its ability to utilize or transform HP.

[0032] 2. Strain identification (1) Morphological identification The purified strain HPD1 was inoculated onto nutrient broth (NA) plates (NA composition: 10.0 g / L peptone, 3.0 g / L beef extract, 5 g / L sodium chloride, 15.0 g / L agar), and cultured at 30°C for 24 h. The colony morphology was then observed.

[0033] The results showed that strain HPD1 was a Gram-negative bacterium that grew rapidly on NA medium, forming distinct colonies within 24 hours. The colonies were approximately 1 mm in diameter, lemon-colored, raised, smooth, and had regular edges. Figure 2 As shown.

[0034] (2) Molecular biological identification: Single colonies were selected, and total DNA was extracted using a bacterial genomic DNA extraction kit (TIANGEN, Beijing, China). The bacterial 16S rRNA gene was amplified by PCR using primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'). The PCR reaction mixture (30 μL) consisted of: 15 μL Premix Taq (Takara, Japan), 0.3 μL each of 10 μM forward and reverse primers, 1 μL template DNA, and 13.4 μL nuclease-free water. The PCR conditions were: 95°C pre-denaturation for 2 min; followed by 30 cycles (95°C denaturation for 30 s, 55°C annealing for 15 s, 72°C extension for 80 s); and a final extension at 72°C for 5 min. Positive and negative controls were included in the reaction. After PCR products were detected by 1% agarose gel electrophoresis, bidirectional Sanger sequencing was performed using a sequencer (Sangon Biotech, Shanghai, China).

[0035] The obtained 16S rRNA gene sequence was aligned with the NCBI database using the BLAST tool (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and the reference sequence with the highest homology was selected and downloaded. Subsequently, multiple sequence alignment was performed using MAFFT (v7.520) software, and the sequences were pruned using the Trimal (v1.4) tool. Based on the pruned alignment results, phylogenetic analysis was performed using IQ-TREE software with 1000 ultrafast bootstrap replicates. The resulting maximum likelihood phylogenetic tree was visualized using the ITOL platform (https: / / itol.embl.de / ). The results are as follows: Figure 3 As shown, strain HPD1 and *Sphingosine monocytogenes* (…) Novosphingobium Species clustering.

[0036] To further clarify its taxonomic position, strain HPD1 was inoculated into Nutrient Broth (NB) and cultured at 30°C with shaking until the logarithmic growth phase. Cells were collected by centrifugation at 4000 r / min for 30 min at 4°C, and genomic DNA was extracted. Subsequently, the PE150 whole genome was sequenced using an Illumina NovaSeq sequencer (Majorbio, Shanghai, China). After quality control and assembly, a draft genome of strain HPD1 was obtained. Using GTDB-Tk (v2.4.0) software, strain HPD1 was annotated based on 120 conserved bacterial marker proteins. Simultaneously, reference genomes of 14 *Neosphingosine* species and their closely related species were downloaded from the NCBI database for comparative analysis. Based on the tandemly conserved protein sequences of all genomes, a maximum likelihood phylogenetic tree was constructed using IQ-TREE software (v2.4.0) and visualized using ITOL. Phylogenetic analysis at the genome level showed (e.g.) Figure 4 As shown), strain HPD1 and Novosphingobium mathurense They are most closely related and have the highest sequence consistency.

[0037] Based on the combined results of 16S rRNA gene sequence analysis and whole-genome phylogenetic analysis, this strain was identified as *Neosphingosine Matura* (…). Novosphingobium mathurense It was named Matura Neosphingosine Hydrochloride HPD1.

[0038] The novel sphingosine monocytogenes HPD1 was deposited on May 9, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38610, located at No. 1 Beichen West Road, Chaoyang District, Beijing.

[0039] Example 3: Preparation of microbial inoculant and analysis of its degradation rate of fluopyram. 1. Preparation of Neosphingosine Matura HPD1 microbial inoculum *Neosphospira matura* HPD1 was inoculated into 100 mL of NB liquid medium and cultured with shaking at 30°C and 120 rpm for 12–16 h to obtain bacterial suspension. The suspension was then aliquoted into 50 mL sterile centrifuge tubes and centrifuged at 4°C and 4000 rpm for 30 min to collect the bacterial cells. The cells were washed three times with PBS buffer to remove residual culture medium components, then resuspended in MSM basal salt medium and OD was adjusted. 600 The standard seed solution, i.e., microbial inoculant, is prepared by reaching 1.0.

[0040] 2. Degradation performance test Microbial inoculum was inoculated at a rate of 5% (v / v) into 100 mL of MSM medium containing *HP* (final concentration 50 mg / L). A control group of MSM medium without bacteria was also included, with three replicates per group. All cultures were incubated at 30°C with shaking at 120 rpm in the dark. Samples were taken at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 16 h, 24 h, 48 h, 72 h, and 120 h, and the residual concentration of *HP* in the culture medium was determined by LC-MS / MS.

[0041] The total degradation rate at each time point is calculated using the following formula:

[0042] Among them, E f The percentage of HP degradation is represented by C0; C0 is the initial concentration of HP in the culture system (mg / L); C t The residual concentration of HP in the experimental group at time t (mg / L) is given.

[0043] The results show (e.g.) Figure 5As shown in the figure, *Neosphomonas matulata* HPD1 exhibited highly efficient degradation capacity for *Helicobacter pylori* (HP) during a 120-hour culture period. In the inoculated treatment group, the initial HP concentration was 50.0 mg / L. Within 0–4 hours, the concentration only decreased slightly, indicating a brief adaptation phase; subsequently, the degradation rate accelerated significantly. By 10 hours, the residual HP concentration decreased to 16.5 mg / L, with a total degradation rate of 67.0%; by 16 hours, the residual concentration further decreased to 1.6 mg / L, with a total degradation rate exceeding 96%; and by 24 hours, the residual HP concentration decreased to 0.4 mg / L, with a total degradation rate exceeding 99%. Thereafter, HP remained at extremely low residual levels in the culture system. In contrast, the HP concentration in the uninoculated control group only slowly decreased from 50.0 mg / L to 45.1 mg / L within 120 hours, without significant degradation. These results demonstrate that *Neosphomonas matulata* HPD1 can rapidly and efficiently degrade HP, showing good potential for bioremediation of HP pollution.

[0044]

[0045] Example 4: Study on the degradation characteristics of high-efficiency fluopyram by microbial agents To systematically evaluate the impact of environmental factors on the ability of microbial agents to degrade HP, this example investigated the effects of pH, culture temperature, and inoculum size on degradation efficiency.

[0046] (1) Effect of pH on the degradation of HP by microbial agents The initial pH of the MSM medium was adjusted to 5.0, 6.0, 7.0, 8.0, and 9.0 by adding appropriate amounts of 100 mM HCl or NaOH. HP (final concentration 50 mg / L) was added to the medium at each pH condition, and the microbial agent prepared in Example 3 was inoculated at a rate of 5% (v / v), with three replicates for each group. All treatments were cultured at 30°C and 120 rpm in the dark with shaking for 120 h. Samples were taken at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 16 h, 24 h, 48 h, 72 h, and 120 h, and the residual concentration of HP was determined by LC-MS / MS.

[0047] The results show (as shown in Table 5 and) Figure 6As shown in the figure, the microbial agent effectively degraded *H. pylori* within a pH range of 5.0–9.0. Specifically, under pH conditions of 6.0–9.0, the *H. pylori* concentration decreased rapidly in the early stages of cultivation, and was essentially completely degraded by 16–24 h. In contrast, the degradation rate was relatively slower at pH 5.0, but *H. pylori* was also essentially completely degraded by 48 h. These results indicate that the *Neosphomonas matulata* HPD1 microbial agent possesses stable *H. pylori* degradation ability within a pH range of 5.0–9.0, and exhibits high degradation activity under neutral to slightly alkaline conditions.

[0048]

[0049] (2) Effect of temperature on the degradation of HP by microbial agents Microbial inoculum was added to 100 mL of MSM medium containing *HP* (final concentration 50 mg / L) at an inoculation rate of 5% (v / v). The medium was then incubated for 120 h in a constant-temperature shaker at 20°C, 25°C, 30°C, 35°C, and 40°C, respectively, in the dark, with three replicates per group. Samples were taken at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 16 h, 24 h, 48 h, 72 h, and 120 h, and the residual concentration of *HP* in the medium was determined using LC-MS / MS.

[0050] The results show (as shown in Table 6 and) Figure 7 As shown in the figure, the microbial inoculant exhibited good degradation ability within the temperature range of 20–35°C. 30°C was the optimal degradation temperature, under which the HP concentration decreased rapidly within 10–16 h and was essentially completely degraded by 24 h. The degradation effect at 25°C was similar to that at 30°C. At 20°C and 35°C, the degradation rate decreased, but HP was still almost completely degraded by 48 h. In contrast, the HP concentration did not decrease significantly during the culture period at 40°C, indicating that excessively high temperatures inhibited the degradation activity of the inoculant. These results indicate that *Neosphomonas matulata* HPD1 exhibits optimal degradation performance in the 25–30°C range, with an optimal temperature of 30°C, demonstrating good mesophilic adaptability and practical application potential.

[0051]

[0052] (3) Effect of microbial inoculum amount on HP degradation rate Microbial inoculum was added to 100 mL of MSM medium containing *HP* (final concentration 50 mg / L) at inoculation rates of 1%, 5%, 10%, and 20% (v / v), respectively. The medium was incubated at 30°C and 120 rpm in the dark with shaking for 120 h, with three replicates per group. Samples were taken at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 16 h, 24 h, 48 h, 72 h, and 120 h, and the residual concentration of *HP* in the medium was determined by LC-MS / MS.

[0053] The results are shown in Table 7 and Figure 8 As shown, the inoculum size of the microbial agent has a significant impact on the degradation rate of *Helicobacter pylori* (HP). Within the inoculum size range of 1%–20% (v / v), the degradation rate of HP increased significantly with increasing inoculum size. Specifically, the 20% inoculum treatment group rapidly reduced the HP concentration to extremely low levels within 6–10 h of culture; the 10% inoculum treatment group showed rapid degradation within 10–16 h; the 5% inoculum treatment group was essentially degraded within 16–24 h; and even at a 1% inoculum size, HP was almost completely degraded within 48 h. In the uninoculated control group, the HP concentration only decreased slightly during the 120 h culture period, with no significant degradation observed.

[0054]

[0055] Example 5: Verification of the degradation of highly efficient flupyradifurone in surface sediments of coastal wetlands by microbial agents To evaluate the actual degradation capacity of microbial agents for *Helicobacter pylori* (HP) in a coastal wetland environment, surface sediments (0–10 cm) and nearshore overlying water were collected from the Chongming Dongtan coastal wetland in Shanghai for a simulated degradation experiment. The collected soil samples were thoroughly mixed and divided into ten groups, with the following treatment settings for each group: Sterilized soil + uninoculated soil (sterilized control); Sterilized soil + 1% microbial inoculum; Sterilized soil + 5% microbial inoculant inoculation; Sterilized soil + 10% microbial inoculant inoculation; Sterilized soil + 20% microbial inoculant inoculation; Unsterilized soil + uninoculated (natural control); Unsterilized soil + 1% microbial inoculum; Unsterilized soil + 5% microbial inoculum; Unsterilized soil + 10% microbial inoculum; Unsterilized soil + 20% microbial inoculum; The specific procedures are as follows: After sterilizing the nearshore overwater under high temperature and pressure (121°C, 20 min), microbial inoculum was added at inoculation rates of 1%, 5%, 10%, and 20% (v / v), respectively; the uninoculated treatment group used an equal volume of sterilized overwater. HP standard solution was added to the overwater of all treatment groups to achieve a final concentration of 50 mg / L. 40 g of soil sample (sterilized soil from the sterilized group) was weighed and placed in a 100 mL breathable glass bottle. Overwater containing or without the bacterial solution was added at a 1:1 (w / v) ratio, and the mixture was thoroughly mixed to form a homogeneous slurry. Each treatment group was set up in triplicate and incubated at 30°C and 120 rpm in the dark with shaking for 120 h. Samples were taken at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 16 h, 24 h, 48 h, 72 h, and 120 h. Before sampling, the culture system was thoroughly shaken and mixed. Then, 500 μL of homogenized mud sample was taken and extracted and purified according to the sample pretreatment method described in Example 1. That is, 400 μg formic acid was added and mixed, then 5 mL acetonitrile was added and vortexed for 3 min. After adding 0.2 g NaCl, vortexing was continued for 3 min. After centrifugation at 6000 r / min for 5 min, the supernatant was filtered through a 0.22 μm organic phase nylon filter membrane, and the residual HP concentration was determined by LC-MS / MS method described in Example 1.

[0056] The results are as follows Figure 9 As shown, in the sterilized (left) and non-sterilized (right) coastal wetland surface sediment systems, the HP concentration in the uninoculated control group decreased only slowly within 120 h, indicating that its natural loss or natural degradation was limited. The natural control group (6) showed a slightly higher decrease than the sterilized control group (1), indicating that the indigenous microorganisms in the sediments had a certain transformation effect on HP, but the degradation capacity was relatively limited. After inoculation with microbial agents, the degradation rate of HP in both sterilized and non-sterilized sediment systems was significantly increased. In the sterilized system, the 20% and 10% inoculation treatment groups reduced HP to extremely low levels in about 8–10 h and 10 h, respectively; the 5% and 1% inoculation treatment groups also achieved efficient degradation within 24–48 h. In the non-sterilized system, the 20% inoculation treatment group had already reduced to a low level by 24 h; the 10% and 5% inoculation treatment groups achieved efficient degradation within 24–48 h; and the residual concentration in the 1% inoculation treatment group was significantly reduced by 120 h.

[0057] Under the same inoculum amount, the degradation rate in the sterilized sediment system was generally faster than that in the non-sterile sediment system, indicating that HPD1 can exert its degradation effect more quickly in an environment without competition or interference from indigenous microorganisms. In the non-sterile sediment system, although the degradation process was somewhat delayed, HPD1 inoculation still significantly promoted HP degradation, indicating that this agent can maintain relatively stable degradation activity in the complex sediment microecological environment.

[0058] Example 6: Product identification and transformation pathway analysis of microbial inoculants degrading high-efficiency flupyradifurone. To analyze the degradation products of *Helicobacter pylori* (HP) by microbial inoculants, a 5% (v / v) inoculum was added to 100 mL of MSM medium containing HP (final concentration 50 mg / L). The medium was incubated at 30°C and 120 rpm in the dark with shaking for 120 h, with three replicates per group. Samples were taken at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 16 h, 20 h, 24 h, 48 h, 72 h, and 120 h. HP and its transformation products were analyzed using a Waters ACQUITY UPLC I-Class / Xevo G3 QTof high-resolution liquid chromatography-mass spectrometry (LC-MS / MS). The chromatographic conditions were as follows: ACQUITY Premier HSS T3 column (1.8 μm, 2.1 mm × 100 mm); column temperature: 40°C; injection volume: 3 μL. Table 8 shows the mobile phase and elution conditions in ESI positive ion mode, and Table 9 shows the mobile phase and elution conditions in ESI negative ion mode.

[0059]

[0060] Mass spectrometry was performed using ESI positive and negative ion modes. The capillary voltage was 1.0 kV, and the cone voltage was 30 V. The ion source temperature was 120°C, the nebulizer gas temperature was 400°C, and the nebulizer gas flow rate was 800 L / h. The acquisition mode was MSE; the collision energies were set to low energy (6 eV) and high energy (15–40 eV). HP and its transformation products were identified based on the retention time, precise molecular mass, isotopic distribution, and secondary mass spectrometry fragmentation information.

[0061] UPLC-QTof high-resolution mass spectrometry analysis revealed three main transformation products during the degradation of HP by *Neosphomonas matulata* HPD1: M196, M305, and M377. Their retention times, monoisotope masses, and molecular formulas are shown in Table 10.

[0062]

[0063] The results are as follows Figure 10As shown, the degradation of *Neosphomonas matulata* HPD1 by HP may involve transformation reactions such as aryl ether bond cleavage, side chain cleavage, and hydroxylation. The response value trends show that the HP parent compound rapidly decreases during cultivation; simultaneously, the response value of M196 rapidly increases and remains at a high level in the later stages, indicating that M196 is the main stable transformation product formed by *Neosphomonas matulata* HPD1 during HP degradation. Based on high-resolution mass spectrometry results, it is speculated that the aryl ether bond in the HP molecule is an important cleavage site, and *Neosphomonas matulata* HPD1 can directly generate M196 through aryl ether bond cleavage. Furthermore, HP can also first undergo hydroxylation to form M377, then undergo side chain cleavage to form M305, followed by further aryl ether bond cleavage to generate M196. These results further demonstrate that HPD1 has a clear HP transformation capacity, providing experimental evidence for its application in the bioremediation of HP residual pollution.

[0064] Example 7: Whole genome analysis of *Neosphomonas matulata* HPD1 To further identify the strain and analyze its metabolic and environmental adaptation potential at the genomic level, whole-genome sequencing was performed on *Neosphomonas matulata* HPD1. After sequence assembly and quality control, the genome sequence of *Neosphomonas matulata* HPD1 was obtained, with a total length of 4,4619.19 bp and a GC content of 63.147%. Genome annotation predicted 4,106 coding sequences (CDS), accounting for 90.11% of the total genome length; 3 rRNA and 46 tRNA genes were also predicted. The overall genome characteristics were visualized using a genome loop diagram, as shown below. Figure 11 As shown.

[0065] (1) COG function annotation After comparing all CDS with the COG database, a total of 3,368 genes were classified into 23 COG functional categories, accounting for 82.03% of the total CDS. Among them, genes related to general function prediction were the most numerous, with 299; followed by genes related to lipid transport and metabolism (289), transcription genes (269), cell wall / membrane / capsule biogenesis genes (263), and energy production and conversion genes (254), such as... Figure 12 As shown in the figure, the enrichment of these functional categories indicates that *Neosphomonas matura* HPD1 possesses a relatively rich collection of genes related to material transport and energy metabolism, providing a genetic basis for its adaptation to complex environments and transformation of exogenous organic pollutants such as *HP*. These results can provide a basis for subsequent screening of candidate genes for *HP* degradation and for elucidating the degradation mechanism.

[0066] (2) GO function annotations Annotation results based on the GO database show that 3025 genes were classified functionally, accounting for 73.67% of the total CDS. In biological processes, the most annotated entries included protein hydrolysis (69) and translation (60). In cellular components, most gene products were located in the membrane (457), cytoplasm (362), plasma membrane (333), and cytoplasm (256). In molecular functions, genes related to ATP binding (255), metal ion binding (223), DNA binding (196), and DNA-binding transcription factor activity (120) were the most abundant. Figure 13 As shown above, these results further corroborate that *Neosphomonas matura* HPD1 possesses active basal metabolism, material transport, energy supply, and gene expression regulation capabilities, providing a genetic basis for its adaptation to complex environments and degradation of exogenous organic pollutants such as *HP*.

[0067] (3) KEGG annotation Functional annotations were obtained for 3013 coding genes by comparing them with the KEGG database. Among them, 1065 genes are involved in various metabolic pathways, accounting for the largest proportion. The rest are mainly involved in environmental information processing (such as two-component systems, 174 genes) and cellular processes (such as quorum sensing, 52 genes). Figure 14 As shown.

[0068] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. *Sphingosine Matura* strain for degrading highly efficient flupyradifurone (SMR) Novosphingobium mathurense HPD1, characterized in that: The *Sphingosine Matura* HPD1 strain was deposited on May 9, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.38610, located at No. 1, Beichen West Road, Chaoyang District, Beijing.

2. A microbial agent for degrading highly effective flupyradifurone, characterized in that: The microbial agent comprises Matura sphingosine monocytogenes HPD1 as described in claim 1 for degrading highly efficient flupyradifurone.

3. The microbial agent for degrading highly efficient flupyradifurone as described in claim 2, characterized in that: The microbial agent can rapidly degrade highly efficient flupyradifurone, with a total degradation rate of over 99%.

4. The microbial agent for degrading highly efficient flupyradifurone as described in claim 2, characterized in that: The pH of the environment in which the microbial agent degrades highly efficient flupyradifurone is 5.0–9.

0.

5. The microbial agent for degrading highly efficient flupyradifurone as described in claim 2, characterized in that: The optimal temperature for the degradation of highly efficient flupyradifurone by the microbial agent is 25–30°C.

6. The microbial agent for degrading highly efficient flupyradifurone as described in claim 2, characterized in that: The degradation rate of highly efficient flupyradifurone by the microbial agent increases with the increase of the inoculum amount.

7. The microbial agent for degrading highly efficient flupyradifurone as described in claim 2, characterized in that: The microbial agent can maintain stable degradation activity in the complex sediment microecological environment.

8. A method for preparing the microbial agent for degrading highly efficient flupyradifurone as described in any one of claims 2 to 7, characterized in that: The *Neosphospira matura* HPD1 strain was inoculated into 100 mL of NB liquid medium and cultured with shaking at 30°C and 120 rpm for 12–16 h to obtain a bacterial suspension. The suspension was then aliquoted into 50 mL sterile centrifuge tubes and centrifuged at 4°C and 4000 rpm for 30 min to collect the bacterial cells. The cells were washed three times with PBS buffer to remove residual culture medium components, then resuspended in basal salt liquid medium and OD was adjusted. 600 The standard seed solution, i.e., the microbial inoculant, is prepared by reaching version 1.0.

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