Rhodococcus pyridinovorans strain for degrading thiamethoxam and application thereof

Abstract

The application belongs to the field of microorganisms and the field of environmental protection, and provides a pyridine-degrading Rhodococcus pyridinovorans and application thereof. The pyridine-degrading Rhodococcus pyridinovorans is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 37890. The application first discovers the Rhodococcus with thiamethoxam-degrading capacity, and the strain has strong degrading performance and environmental adaptation capacity; in the treatment of thiamethoxam pollution in soil and water, the application has clear applicability and popularization value.

Description

Technical Field

[0001] This application pertains to the fields of microbiology and environmental protection. Specifically, this application provides a strain of Rhodococcus pyridostigmine that degrades thiamethoxam and its applications. Background Technology

[0002] Thiamethoxam, a representative second-generation neonicotinoid insecticide, is widely used for pest control in various crops such as rice, vegetables, and fruit trees due to its highly effective and broad-spectrum systemic insecticidal activity. However, thiamethoxam has high water solubility (4.1 g / L), relatively stable chemical properties, and easily enters water bodies through agricultural runoff and persists in soil, posing a serious sublethal toxicity threat to non-target pollinating insects such as bees, and may accumulate through the food chain, ultimately endangering human health. Therefore, developing efficient and environmentally friendly thiamethoxam residue reduction technologies has become a research hotspot in the field of environmental science.

[0003] Currently, methods for removing thiamethoxam residues from the environment mainly include physical adsorption, chemical oxidation, and bioremediation. While physicochemical methods can rapidly remove pollutants, they suffer from drawbacks such as high cost, potential for secondary pollution, and significant disturbance to the in-situ ecosystem. Bioremediation technology has attracted considerable attention due to its low cost and good ecological compatibility. In recent years, researchers have isolated several microorganisms with thiamethoxam-degrading capabilities from different habitats. Chinese patent CN120081507A discloses the application of a nitrogen-fixing cyanobacterium in degrading thiamethoxam in water, involving the complete degradation of 10 mg / L thiamethoxam by the nitrogen-fixing cyanobacterium (Nostoc sp. PCC7120) within 8 days. Furthermore, Chinese patent CN110106116A discloses a strain of *Enterobacter cloacae* (CGMCC No. 16235), which has been confirmed to have a thiamethoxam-degrading effect. Despite the discovery of these microbial resources, the diversity of thiamethoxam-degrading strains remains insufficient.

[0004] Although strains of the genus Rhodococcus have been found capable of degrading various pesticides, there are currently no reports on the use of Rhodococcus to degrade the neonicotinoid insecticide thiamethoxam. Existing thiamethoxam-degrading strains still face challenges in practical applications, including degradation efficiency being greatly affected by environmental factors, unclear degradation mechanisms, and insufficient adaptability to complex pollution scenarios. Summary of the Invention

[0005] On the one hand, this application provides a strain of Rhodococcus pyridinivorans that degrades thiamethoxam. The Rhodococcus pyridinivorans is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 37890.

[0006] On the other hand, this application provides a microbial agent containing the aforementioned Rhodococcus pyridostigmine.

[0007] Furthermore, the bacterial agent is a solid bacterial agent or a liquid bacterial agent.

[0008] Furthermore, the microbial agent also contains excipients.

[0009] On the other hand, this application provides the application of the aforementioned Rhodococcus pyridostigmine or its agent in the degradation of neonicotinoid insecticides in the environment.

[0010] Furthermore, the neonicotinoid insecticide is thiamethoxam.

[0011] Furthermore, the environment described is an aquatic environment.

[0012] Furthermore, the environment described is a soil environment.

[0013] On the other hand, this application provides the application of the above-mentioned Rhodococcus pyridostigmine or its agent in the preparation of neonicotinoid insecticide degrading agents.

[0014] Furthermore, the neonicotinoid insecticide degrading agent is a thiamethoxam degrading agent.

[0015] Beneficial effects:

[0016] The *Rhodococcus* strain described in this invention can grow using thiamethoxam as its sole carbon source and exhibits highly efficient degradation capabilities for thiamethoxam. In inorganic salt culture medium, low-concentration thiamethoxam (5-10 mg / L) achieves complete degradation on day 14; the degradation rate reaches 70% at medium concentration (50 mg / L). This strain shows good tolerance to thiamethoxam, with degradation rates of 48% and 39% at high concentrations (100 and 200 mg / L), respectively. Compared with existing technologies, the *Rhodococcus* strain of this invention has strong environmental adaptability and demonstrates the potential to degrade various pollutants. It has clear applicability and promotional value in the remediation of thiamethoxam pollution in soil and water bodies, providing important strain resources and technical support for functional microbial enhancement technologies. Attached Figure Description

[0017] Figure 1 The morphological characteristics of strain D are shown (the left side shows the colony appearance, and the right side shows the scanning electron microscope image).

[0018] Figure 2 This is the phylogenetic tree of strain D.

[0019] Figure 3 This study demonstrates the degradation performance of thiamethoxam by Rhodococcus at different substrate concentrations.

[0020] Figure 4 This demonstrates the degradation performance of thiamethoxam by Rhodococcus under different inoculum levels.

[0021] Figure 5 This demonstrates the degradation performance of thiamethoxam by Rhodococcus at different pH values. Detailed Implementation

[0022] The culture medium involved in the embodiments includes:

[0023] Beef extract peptone (LB) medium: 3g beef extract, 5g NaCl, 10g peptone, 1L distilled water, 20g agar, pH 7.

[0024] Inorganic salt (MSM) medium: KH2PO4 0.9 g, Na2HPO4 6.5 g, MgSO4·H2O 0.2 g, NaCl 1.6 g, distilled water 1 g, pH 7.0.

[0025] Example 1: Isolation, screening and identification of bacterial strains

[0026] From soil samples contaminated with thiamethoxam, 5.00 g of soil sample was added to an inorganic salt medium (MSM) containing 10 mg / L thiamethoxam and cultured in a constant temperature shaker (30℃, 160 rpm) for 7 days. After 7 days, the sample was transferred at a 5% inoculum (V / V) to an MSM medium containing 50 mg / L thiamethoxam and cultured again under the same conditions for 7 days. This process was repeated, and the sample was then transferred to an MSM medium containing 100 mg / L thiamethoxam for a total of 3 consecutive enrichment cultures. Samples were taken 14 days after the end of the culture period to determine the degradation rate of thiamethoxam in the medium. Samples showing degradation were diluted to 10... -4 10 -5 10 -6 10 -7 The diluted solution was spread onto LB solid medium and incubated in a constant temperature incubator (26℃) for 2-5 days. After colonies appeared on the medium, they were picked and inoculated onto LB plates. Single colonies were isolated by the streak plate method. The obtained single colonies are shown in the figure. Figure 1 As shown.

[0027] The degrading strain was inoculated onto LB solid medium and cultured in an incubator for 2–3 days. Single colony morphology was observed visually. Then, test strains were picked and cultured in LB liquid medium until the logarithmic growth phase. Cells were collected by brief, slight centrifugation. Freshly prepared phosphate buffer was added, and the cells were centrifuged at 1000 rpm for 5 min at low temperature, discarding the supernatant. The cells were resuspended again with fresh buffer for 15 min, centrifuged at 1000 rpm for 5 min at low temperature, and the supernatant was discarded. This process was repeated twice, collecting cells the size of mung beans. Fresh glutaraldehyde fixative was then slowly added to EP tubes, and the cells were fixed overnight at 4°C. The fixed samples were washed three times with phosphate buffer, followed by gradient dehydration using ethanol solutions of different concentrations (30%, 50%, 70%, 80%, 90%). After dehydration, tert-butanol was used for replacement, and the samples were air-dried. Finally, the samples were sputter-coated with gold, and the morphology of the cells was observed under a scanning electron microscope. The resulting electron microscope images are shown below. Figure 1 As shown.

[0028] The bacterial strains were identified using a combination of physiological and biochemical characteristics and 16S rRNA sequence. Colony PCR primers were used: 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1) and 1492R (5'-GGTTACCTTGTTACGAC-3', SEQ ID NO.2). The 16S rRNA gene fragment was amplified using these primers and sequenced. The obtained sequence was preliminarily identified as a strain by BLAST alignment using NCBI. In this example, the strain was identified as *Rhodococcus pyridinivorans*. Figure 2 As shown.

[0029] On March 22, 2026, the strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its accession number is CGMCC No. 37890, and it is classified as Rhodococcus pyridinivorans.

[0030] Example 2 Degradation characteristics

[0031] Different substrate concentrations:

[0032] A 5% inoculum suspension was inoculated into 50 mL of sterile MSM liquid medium at pH 7. Thiamethoxam was added at concentrations of 5 mg / L, 10 mg / L, 50 mg / L, 100 mg / L, and 200 mg / L, respectively. The mixture was thoroughly mixed and incubated in a constant-temperature shaker at 160 rpm in the dark. A blank control group without the bacterial suspension was also included. Each treatment was performed in triplicate. Samples were taken on days 1, 3, 5, 7, 10, and 14 of incubation, and the degradation rate was determined using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS).

[0033] The results are as follows Figure 3 As shown, with increasing culture time, the degradation rate of thiamethoxam by the strain gradually increased within the concentration range of 10–200 mg / L. The low-concentration group (5–10 mg / L) achieved complete degradation by day 14; the medium-concentration group (50 mg / L) achieved a degradation rate of 70%; while the high-concentration groups (100 and 200 mg / L) showed a significant decrease in degradation performance, dropping to 48% and 39%, respectively.

[0034] Different inoculum amounts:

[0035] The bacterial suspension was inoculated into 50 mL of sterile MSM liquid medium (pH 7) containing 50 mg / L thiamethoxam at inoculation rates of 1%, 3%, 5%, 7%, and 9%, respectively. After thorough mixing, the medium was incubated in a constant-temperature shaker at 160 rpm in the dark. A blank control without bacterial suspension was included. Each treatment was performed in triplicate. Samples were collected at 1, 3, 5, 7, 10, and 14 days, and the degradation rate was determined using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS).

[0036] The results are as follows Figure 4 As shown, the degradation efficiency of strain D increased progressively with the inoculum concentration from 3% to 9%. After 14 days of culture, the degradation rates of thiamethoxam in the inoculum concentration groups (3%, 5%, 7%, and 9%) reached 55%, 58%, 57%, and 64%, respectively. Significance analysis of the degradation rate on day 14 revealed no significant differences among the 3%, 5%, 7%, and 9% inoculum concentration groups (P>0.05).

[0037] Different pH values:

[0038] The bacterial suspension was added at a 5% inoculum to 50 mL of sterile MSM liquid medium containing 50 mg / L thiamethoxam. The pH of the medium was adjusted to 5, 6, 7, 9, and 10, respectively, and the medium was incubated in a constant temperature shaker at 160 rpm in the dark. A blank control without bacterial suspension was also included, and each treatment was performed in triplicate. Samples were taken at 1, 3, 5, 7, 10, and 14 days, and the degradation rate was determined using ultra-high performance liquid chromatography-tandem mass spectrometry.

[0039] The results are as follows Figure 5As shown, in the pH gradient experiment, the neutral environment (pH=7) exhibited the strongest degradation activity, with a degradation rate of 73% after 14 days. Under alkaline conditions (pH=9 and 10), the strain maintained high metabolic capacity, with degradation rates of 67% and 66%, respectively. This may be related to its alkali tolerance or the pH tolerance range of the degradation-related enzyme system. However, acidic environments (pH≤6) significantly inhibited the activity of the strain, with the degradation system almost inactivated at pH=5, and a degradation rate of only 16% after 14 days.

[0040] Example 3 Degradation spectrum

[0041] A 5% inoculum suspension was inoculated into 50 mL of sterile MSM liquid medium. Imidacloprid, thiamethoxam, and acetamiprid were added at 10 mg / L respectively, and the mixture was thoroughly mixed and incubated in a constant-temperature shaker at 160 rpm in the dark. A blank control group without bacterial suspension was also included. Each treatment was performed in triplicate. Samples were taken on day 14 of incubation, and the degradation rate of different substrates by Rhodococcus was determined using ultra-high performance liquid chromatography-tandem mass spectrometry.

[0042] The results showed that *Rhodococcus pyridinivorans* strain DB_1 had no significant degradation ability against imidacloprid, thiamethoxam, and acetamiprid, but exhibited significant substrate specificity in its degradation of thiamethoxam. Based on the strain's species name "pyridinivorans" and its known pyridine degradation ability, it is speculated that this strain may recognize specific structures (such as nitrogen-containing heterocycles) in the thiamethoxam molecule through a specific metabolic pathway, thus being unable to metabolize other neonicotinoid pesticides with significantly different structures.

Claims

1. A strain of Rhodococcus pyridinivorans that degrades thiamethoxam, characterized in that, The Rhodococcus species is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 37890.

2. A bacterial agent, characterized in that, The bacterial agent contains Rhodococcus pyridostigmine as described in claim 1.

3. The microbial agent according to claim 2, wherein the microbial agent is a solid microbial agent or a liquid microbial agent.

4. The microbial agent according to claim 3, wherein the microbial agent further comprises excipients.

5. The application of the Rhodococcus pyridostigmine according to claim 1 or the bacterial agent according to claim 2 in the degradation of neonicotinoid insecticides in the environment.

6. In the application according to claim 5, the neonicotinoid insecticide is thiamethoxam.

7. The application according to claim 6, wherein the environment is an aquatic environment.

8. The application according to claim 6, wherein the environment is a soil environment.

9. The application of the Rhodococcus pyridostigmine according to claim 1 or the bacterial agent according to claim 2 in the preparation of neonicotinoid insecticide degrading agents.

10. In the application according to claim 9, the neonicotinoid insecticide degrading agent is a thiamethoxam degrading agent.

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