Rhodococcus pyridinovorans capable of degrading indoxacarb and application thereof

Abstract

The application discloses a pyrrolnitrin-degrading Rhodococcus pyridinovorans and application thereof, wherein the Rhodococcus pyridinovorans is preserved in the China General Microbiological Culture Collection Center, the preservation date is January 14, 2026, the preservation number is CGMCC NO: 37394, and the Rhodococcus pyridinovorans can efficiently degrade the pesticide indoxacarb. The application also studies the optimal temperature, pH and inoculation amount and the like of the Rhodococcus pyridinovorans for degrading indoxacarb. The Rhodococcus pyridinovorans enriches germplasm resources of indoxacarb-degrading bacteria, provides efficient functional strains for bioremediation of indoxacarb-contaminated soil, and has great practical application value.

Description

Technical Field

[0001] This invention belongs to the field of microbial degradation and soil bioremediation technology, specifically relating to a pyridine-eating Rhodococcus faecalis that degrades indoxacarb and its application. Background Technology

[0002] Indoxacarb belongs to the diazinon class of organic insecticides. It is a novel insecticide developed by DuPont in the late 20th century and was the first commercially available sodium channel blocker. Its chemical formula is C0.05. 22 H 17 ClF3N3O7 has a molecular weight of 527.84 and a density of 1.53 g / cm³. 3 It has a flash point of 299.3℃, and common formulations are 30% water-dispersible granules and 15% suspension concentrates. As a typical representative of this type of insecticide, its core characteristic is its unique target of action and the absence of cross-resistance with traditional insecticides such as organophosphates and pyrethroids, making it an important variety in integrated pest management and resistance control. Regarding its mechanism of action, indoxacarb itself has relatively weak insecticidal activity. After entering the insect's body, it is rapidly converted into the metabolite DCJW (N-2-demethoxycarbonyl metabolite). This metabolite specifically binds to the inactivated state of voltage-gated sodium channels in nerve cells, irreversibly inhibiting sodium ion influx, blocking nerve signal transmission, leading to insect motility disorder, cessation of feeding, and ultimately paralysis and death.

[0003] Indoxacarb, a widely used pesticide, poses a significant risk to the ecological environment and biological health due to its residue problems. Studies have shown that the average concentration of this pesticide in surface water ranges from 3.7 to 7.8 μg / L, with a maximum concentration reaching 13.7 μg / L. In agricultural soils such as cotton, rice, and cabbage fields, its degradation half-life is approximately 7–11 days. Toxicological data show that indoxacarb has significant toxic effects on rats during their growth and development stages, primarily damaging spleen and liver function. Its toxicity to non-target organisms such as bees, zebrafish, and silkworms is classified as moderate to high, with zebrafish exhibiting genotoxic characteristics such as hepatocyte apoptosis and DNA damage after exposure. Due to high application rates or improper handling, indoxacarb residues are widely present in crops, water bodies, and soil environments. Long-term accumulation not only disrupts the balance of soil microbial communities but also poses a potential threat to ecosystem health and human safety. Therefore, developing efficient and feasible technologies for eliminating indoxacarb environmental residues has become an urgent and practically significant research task in the fields of environmental science and agriculture.

[0004] Bioremediation, as an emerging environmental governance strategy, primarily relies on the decomposition of pollutants by microorganisms or their metabolites, ultimately transforming the pollutants into harmless inorganic small molecules. This technology boasts advantages such as high efficiency, safety, no residue, and no secondary pollution, and is gradually becoming a preferred solution for addressing various environmental pollution problems, including pesticide residues and heavy metal contamination.

[0005] In the prior art, researchers have isolated some microorganisms with the potential to degrade indoxacarb from different environments. For example, patent document CN202411296737.2 discloses a chiral Bacillus abhattai that degrades indoxacarb enantiomers and its application. Specifically, it discloses that Bacillus abhattai isolated from a wastewater treatment system exhibits degradation activity against indoxacarb, and focuses on its selective degradation behavior of chiral enantiomers. Patent document CN202410068596.2 discloses *Rhodococcus pyridostigma*, its microbial agent, and their application in the remediation of petroleum hydrocarbon-contaminated soil. Specifically, it discloses that *Rhodococcus pyridostigma* has a significant removal effect on petroleum hydrocarbons in soil. A biomass containing this *Rhodococcus pyridostigma* achieved a petroleum hydrocarbon degradation rate of 63.25% after 14 days, and after adding a carbon source, the degradation rate reached 82.21% after 14 days. Furthermore, this *Rhodococcus pyridostigma* can withstand 5000 mg / kg of petroleum hydrocarbons. This *Rhodococcus pyridostigma* and the microbial agent made from the strain can be used for biomass treatment of soil contaminated with high concentrations of petroleum hydrocarbons. Patent document CN201310275215.X discloses the application of a strain of *Rhodococcus pyridostigma* in the degradation of zearalenone. *Rhodococcus pyridostigma* is a Gram-positive bacterium that can obtain metabolites with significant degradation effects on zearalenone through microbial fermentation, showing promising development prospects as a biological detoxifier for fungal toxins. However, in practical environmental remediation applications, besides selectivity for specific enantiomers, the overall environmental adaptability and operational stability of degrading strains are often the key bottlenecks determining their remediation efficacy. Currently, there is a lack of systematic research and high-performance strain resources regarding the physiological characteristics of degrading strains that directly affect their field application effects on soil, such as their ability to maintain activity across a wide pH and temperature range and their tolerance to high-concentration pesticide shock loads. Furthermore, there are no reports on the degradation of indoxacarb by *Rhodococcus pyridinivorans*. Discovering new functional strains of this genus and elucidating their highly efficient degradation characteristics under different environmental conditions is of great significance for expanding the germplasm resource bank of degrading bacteria and developing bioremediation agents adapted to complex environmental conditions. Summary of the Invention

[0006] The purpose of this invention is to provide a *Rhodococcus pyrithioides* strain that degrades indoxacarb and its applications. This *Rhodococcus pyrithioides* strain can efficiently degrade the pesticide indoxacarb. This invention also investigated the optimal temperature, pH, and inoculum size for this *Rhodococcus pyrithioides* strain to degrade indoxacarb. This *Rhodococcus pyrithioides* strain enriches the germplasm resources of indoxacarb-degrading bacteria and provides a highly efficient functional strain for the bioremediation of indoxacarb-contaminated soil, possessing significant practical application value.

[0007] To achieve the above objectives, the present invention employs the following technical solution: a pyridinivoric Rhodococcus pyridinivorans strain that degrades indoxacarb. This pyridinivoran strain is classified and named Rhodococcus pyridinivorans and is deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 14, 2026, with accession number CGMCCNO: 37394.

[0008] The application of Rhodococcus pyridostigmatae in the degradation of indoxacarb, as described in this invention.

[0009] Furthermore, the *Rhodococcus pyridae* can degrade indoxacarb at concentrations below 100 mg / L within a wide temperature range of pH 5-9 and 20-30°C.

[0010] Furthermore, the *Rhodococcus pyridae* strain achieved a 77% degradation rate of 10 mg / L indoxacarb in a basal salt medium with indoxacarb as the sole carbon source.

[0011] The present invention has the following advantages and beneficial effects:

[0012] (1) This invention screened and obtained a strain of Rhodococcus pyridinivorans DPX-f2 that can rapidly degrade indoxacarb. This Rhodococcus pyridinivorans was obtained from soil near a pesticide factory through artificial enrichment culture, isolation and purification, and has a high degradation efficiency for indoxacarb. The Rhodococcus pyridinivorans DPX-f2 achieved a degradation rate of 77% for indoxacarb at a concentration of 10 mg / L in a basal salt medium with indoxacarb as the sole carbon source.

[0013] (2) This invention enriches the germplasm resource bank of indoxacarb-degrading bacteria, provides efficient functional strains for the bioremediation of soil contaminated by indoxacarb residues, and is expected to break through the technical bottleneck of existing pesticide residue pollution control, and has significant practical application value.

[0014] Figure and Table Description

[0015] Figure 1 Colony morphology of Rhodococcus pyridococcus DPX-f2.

[0016] Figure 2Gram staining image of Rhodococcus pyridococcus DPX-f2.

[0017] Figure 3 Scanning electron microscope image of Rhodococcus pyridococcus DPX-f2.

[0018] Figure 4 This is a phylogenetic tree of the 16S rDNA of Rhodococcus pyridococcus DPX-f2 in Example 1.

[0019] Figure 5 The results of the degradation experiment of indoxacarb by Rhodococcus pyridostigma DPX-f2 in Example 2 are shown.

[0020] Figure 6 The results of the degradation characteristics of indoxacarb by Rhodococcus pyridostigma DPX-f2 in Example 3 are shown. Detailed Implementation

[0021] The embodiments of the present invention will be described in detail below with reference to specific examples. These embodiments are provided to more clearly illustrate the technical solutions of the present invention and do not constitute a limitation thereof. Unless otherwise expressly stated, the reagents, methods, and equipment used in the present invention are all of the types conventionally used in this technical field.

[0022] The culture medium formulations involved in the following examples are as follows:

[0023] LB solid medium (g / L): tryptone 10, yeast extract 5, sodium chloride 10, agar powder 15, distilled water 1000 mL, pH 7.2±0.2, dispensed into Erlenmeyer flasks and sterilized (121℃, 20 min) for later use.

[0024] R2A liquid culture medium (g / L): tryptone 0.25, acid-hydrolyzed casein 0.5, yeast extract 0.5, soluble starch 0.5, dipotassium hydrogen phosphate 0.3, magnesium sulfate 0.1, sodium pyruvate 0.3, peptone 0.25, glucose 0.5, distilled water 1000 mL, pH 7.2±0.2. Dispense into Erlenmeyer flasks and sterilize (121℃, 20 min) for later use.

[0025] R2A agar medium (g / L): yeast extract 0.5, peptone 0.5, casein hydrolysate 0.5, glucose 0.5, soluble starch 0.5, dipotassium hydrogen phosphate 0.3, anhydrous magnesium sulfate 0.024, sodium pyruvate 0.3, agar 15.0, distilled water 1000mL, pH 7.2±0.2. Dispense into Erlenmeyer flasks and sterilize (121℃, 20 min) for later use.

[0026] Inorganic salt medium (MSM): Weigh 0.2 g MgSO4·7H2O, 1.5 g K2HPO4, 0.5 g KH2PO4, and 1.0 g NaCl into an appropriate amount of ddH2O until completely dissolved. Adjust the pH to 7.0 and bring the volume to 1 L. Autoclave at 121℃ for 20 min, cool, and store at room temperature for later use. For solid medium, add 2 wt% agar powder.

[0027] Example 1

[0028] Isolation and identification of indoxacarb-degrading strains:

[0029] 1. Screening and isolation of indoxacarb-degrading strains

[0030] 10 g of soil sample was weighed and added to 50 mL of MSM liquid medium containing 10 mg / L indoxacarb. After shaking culture at 30℃ and 180 r / min for 7 days, 5% (V / V) of the previous culture solution was inoculated into new MSM medium, and the indoxacarb concentration in the medium was set at 10 mg / L for continuous subculturing. Then, the culture solution with 5 subcultures was serially diluted and plated on inorganic salt solid plates containing 20 mg / L indoxacarb, and incubated upside down at 30℃ for 1-2 days. After single colonies grew on the plates, single colonies were picked and streaked multiple times on LB solid medium for purification to obtain single bacteria. Subsequently, single bacterial cultures were performed, and the indoxacarb degradation effect was measured.

[0031] Finally, through continuous subculturing, the fifth-generation enrichment solution demonstrated indoxacarb degradation. After 7 days of culture, it showed an 83% degradation rate for 10 mg / L indoxacarb, with product accumulation, indicating that the enrichment solution may contain a functional strain capable of degrading indoxacarb. Through multiple streak purification and degradation verification tests, a highly efficient indoxacarb-degrading strain, designated DPX-f2, was successfully isolated from the fifth-generation enrichment solution and stored at -80°C using 25 wt% glycerol.

[0032] 2. Identification of strains

[0033] (1) Morphological identification

[0034] After inoculating strain DPX-f2 onto LB agar plates and incubating them upside down at 30°C for 48 h, the colony morphology was observed as follows: Figure 1 As shown: On LB agar plates, single colonies of strain DPX-f2 are round, approximately 0.2–0.8 mm in diameter, with smooth, even edges, free of serrations, wrinkles, or fuzzy edges. The colony body is pale orange-pink, with obvious pigment production. After Gram staining, strain DPX-f2 turns blue-purple, indicating it is a Gram-positive bacterium. Figure 2 As shown. And its morphology under a scanning electron microscope is as follows. Figure 3As shown: the bacterial cells are short rod-shaped (0.8 ~ 1.2 μm × 0.3 ~ 0.5 μm), with blunt and rounded ends, without sharp protrusions, curves, or branching. The cell wall surface is smooth and flat, without obvious wrinkles or nodular protrusions. No appendages such as long flagella, pili, spores, or extracellular thick capsules were observed, and the cell morphology is uniform.

[0035] (2) Physiological and biochemical identification

[0036] The physiological and biochemical characteristics of strain DPX-f2 were determined according to the "Handbook of Systematic Identification of Common Bacteria," including the methyl red test, Kovacs indole test, VP test, and starch hydrolysis test. Strain DPX-f2 was negative in the methyl red test, positive in the Kovacs indole test, negative in the VP test, and negative in the starch hydrolysis test.

[0037] (3) Molecular biological identification of 16S rDNA

[0038] Using the total DNA of strain DPX-f2 as a template, the 16S rRNA gene sequence of strain DPX-f2 was amplified using universal primers 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT) and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0039] Sequencing results showed that the 16S rRNA gene sequence of strain DPX-f2 was 1457 bp in length. Comparison with the NCBI database showed that strain DPX-f2 shared 99.93% homology with *Rhodococcus* sp. The phylogenetic tree constructed from the 16S rRNA gene sequences of 10 standard strains of *Rhodococcus* from the comparison results is shown below. Figure 4 As shown. Based on the morphological, physiological and biochemical characteristics of strain DPX-f2 and the analysis of its 16S rRNA gene sequence, strain DPX-f2 was identified as Rhodococcus pyridinivorans.

[0040] Based on the above identification results, the strain was named Rhodococcus pyridinivorans DPX-f2 and deposited at the China General Microbiological Culture Collection Center on January 14, 2026, with accession number CGMCC NO: 37394 and deposit address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0041] Example 2

[0042] The degradation effect of Rhodococcus pyridostigma DPX-f2 on indoxacarb:

[0043] I. Seed Liquid Preparation

[0044] Rhodococcus pyridostigmine DPX-f2 was inoculated into 50 mL of R2A liquid medium and cultured with shaking at 30 °C and 180 r / min until the logarithmic growth phase. The cells were collected by centrifugation at 5000 r / min for 5 min. The cells were washed three times with 50 mL of 0.9% physiological saline. After washing, the supernatant was discarded, and the cells were resuspended in an appropriate amount of 0.9% physiological saline to adjust the OD value. 600 Value is 1.0, reserved.

[0045] 2. Degradation performance test

[0046] The degradation effect of Rhodococcus pyridostigma DPX-f2 on indoxacarb was determined daily over 7 days.

[0047] OD 600 A bacterial culture with a concentration of 1.0 was inoculated into 50 mL centrifuge tubes at a 10% (v / v) inoculum size and thoroughly mixed with inorganic salt liquid culture medium to form a reaction system with a total volume of 5 mL. The initial concentration of indoxacarb in the system was 10 mg / L. A control without inoculation was used, with three replicates for each group, for a total of 7 groups. The mixture was incubated at 30℃ and 180 r / min in a constant temperature shaker for 1–7 days. Acetonitrile was added and the mixture was thoroughly extracted. The upper layer was filtered through a 0.22 μm filter membrane, and the concentration of indoxacarb was determined by HPLC.

[0048] 3. Testing conditions

[0049] Instrument: Waters E2695;

[0050] Column: DIKMA C18 (250 × 4.6 mm)

[0051] Flow rate: 1 mL / min;

[0052] Column temperature: 30 ± 5℃;

[0053] Mobile phase: Acetonitrile: Water = 70:30 (V / V);

[0054] Detection wavelength: 310 nm.

[0055] The degradation rate of indoxacarb is calculated using the following formula:

[0056] Degradation rate (%) = (C0-C1) / C0×100%; C0 is the indoxacarb residue concentration in the control group, and C1 is the indoxacarb residue concentration in the experimental group.

[0057] II. Experimental Results

[0058] Depend on Figure 5The degradation rate of 10 mg / L indoxacarb by Rhodococcus pyridostigma DPX-f2 reached 77% within 7 days. The degradation rate showed a gradually increasing trend within 7 days, while the OD of the bacterial culture in the MSM system also showed a decreasing trend. 600 The values ​​also showed a synchronous upward trend.

[0059] Example 3

[0060] Degradation characteristics of indoxacarb by Rhodococcus pyridostigma DPX-f2:

[0061] I. Experimental Methods

[0062] 1. Effect of initial pH on the degradation of indoxacarb by Rhodococcus pyridostigma DPX-f2

[0063] Indoxacarb to a final concentration of 10 mg / L was added to 4.5 mL of MSM with initial pH values ​​of 3.0, 5.0, 7.0, and 9.0, followed by 0.5 mL of bacterial suspension. The mixture was incubated at 30°C with shaking. After 4 days, samples were taken and extracted with an equal volume of acetonitrile. A control group with 0.5 mL of sterile water was included at each pH. All treatments were performed in triplicate.

[0064] 2. Effect of temperature on the degradation of indoxacarb by Rhodococcus pyridostigma DPX-f2

[0065] Add indoxacarb to 4.5 mL of MSM to a final concentration of 10 mg / L, then add 0.5 mL of bacterial suspension. Incubate at 10℃, 20℃, and 30℃ with shaking. After 4 days, collect samples and extract with an equal volume of acetonitrile. A control group with 0.5 mL of sterile water was included at each temperature. All treatments were performed in triplicate.

[0066] 3. Effect of initial inoculum size on the degradation of indoxacarb by Rhodococcus pyridostigma DPX-f2

[0067] Five treatment groups were set up: 1%, 3%, 5%, 10%, and 20% (V / V). Indoxacarb to a final concentration of 10 mg / L was added to different volumes of MSM, along with the corresponding bacterial suspension. The mixtures were incubated at 30°C with shaking. After 4 days, samples were taken, and an equal volume of acetonitrile was added for extraction. A control group was used without the addition of bacterial suspension. All treatments were performed in triplicate.

[0068] 4. Effect of substrate concentration on the degradation of indoxacarb by Rhodococcus pyridostigma DPX-f2

[0069] Six treatments were set up with indoxacarb concentrations of 10 mg / L, 20 mg / L, 30 mg / L, 50 mg / L, 70 mg / L, and 100 mg / L. Indoxacarb was added to 4.5 mL of MSM at final concentrations of 10 mg / L, 20 mg / L, 30 mg / L, 50 mg / L, 70 mg / L, and 100 mg / L, respectively. Then, 0.5 mL of bacterial suspension was added to each treatment. The mixtures were incubated at 30°C with shaking. Samples were taken after 4 days, and an equal volume of acetonitrile was added for extraction. A control group with 0.5 mL of sterile water was included for each substrate concentration. All treatments were performed in triplicate.

[0070] II. Experimental Results

[0071] 1. The effect of different initial pH values ​​on the degradation of indoxacarb by Rhodococcus pyridostigma DPX-f2 at an inoculum size of 10% (V / V) and a temperature of 30℃. Figure 6 As shown in (a), when the initial pH was 3.0, 5.0, 7.0, and 9.0, the degradation rates of 10 mg / L indoxacarb by *Rhodococcus pyridostigmata* DPX-f2 within 4 days were 10.6%, 44.3%, 45.6%, and 62%, respectively. Overall, the degradation rate increased with increasing pH, reaching its highest value at pH 9.0. There was no significant difference in the degradation rate of indoxacarb by *Rhodococcus pyridostigmata* DPX-f2 at pH 5.0 and 7.0, but both were significantly lower than the pH 9.0 treatment group, indicating that this strain has indoxacarb degradation activity over a wide pH range of 5.0–9.0, with the optimal degradation effect under weakly alkaline conditions (pH 9.0).

[0072] 2. The effect of different temperatures on the degradation of indoxacarb by Rhodococcus pyridostigma DPX-f2 under conditions of 10% (V / V) inoculum and pH 7.0 is as follows: Figure 6 As shown in Figure (b), the degradation rates of indoxacarb by *Rhodococcus pyridostigmatae* DPX-f2 after 4 days were 2.3%, 28%, and 45% at temperatures of 10℃, 28.3℃, and 30℃, respectively. Within this temperature range, the degradation rate of indoxacarb by *Rhodococcus pyridostigmatae* DPX-f2 showed an increasing trend with increasing temperature. The highest degradation rate of indoxacarb by *Rhodococcus pyridostigmatae* DPX-f2 was observed at 30℃, indicating that the optimal temperature for the degradation of indoxacarb by *Rhodococcus pyridostigmatae* DPX-f2 is 30℃.

[0073] 3. The effect of different inoculum sizes on the degradation of indoxacarb by Rhodococcus pyridostigma DPX-f2 under conditions of 30℃ and pH 7.0 is as follows: Figure 6As shown in (c), under inoculum concentrations of 1%, 3%, 5%, 10%, and 20% (V / V), the degradation rates of indoxacarb by *Rhodococcus pyridostigmata* DPX-f2 within 4 days were 54.9%, 60.4%, 63.3%, 65.2%, and 59.8%, respectively. With increasing inoculum concentration, the degradation rate of indoxacarb by *Rhodococcus pyridostigmata* DPX-f2 showed a trend of first increasing and then decreasing. There was no significant difference in the degradation rate of indoxacarb by *Rhodococcus pyridostigmata* at inoxacarb concentrations of 5% and 10%, indicating that *Rhodococcus pyridostigmata* DPX-f2 reached saturation at an inoculum concentration of 5%, and the optimal inoculum concentration for the degradation of indoxacarb by *Rhodococcus pyridostigmata* DPX-f2 was 5%.

[0074] 4. The effect of the initial concentration of indoxacarb on the degradation of indoxacarb by Rhodococcus pyridostigma under the conditions of 30℃ and pH 7.0 is as follows: Figure 6 As shown in Figure (d), under the treatment conditions of 10 mg / L, 20 mg / L, 30 mg / L, 50 mg / L, 70 mg / L and 100 mg / L, the degradation rates of indoxacarb by Rhodococcus pyridostigma DPX-f2 within 4 days were 45.6%, 29.6%, 24.3%, 22%, 14% and 3.3%, respectively. With the increase of substrate concentration, the degradation rate of indoxacarb by Rhodococcus pyridostigma DPX-f2 showed a decreasing trend. The highest degradation rate of indoxacarb by Rhodococcus pyridostigma DPX-f2 was achieved at a substrate concentration of 10 mg / L.

[0075] In summary, this invention screened and obtained a strain of Rhodococcus pyridinivorans DPX-f2 that can rapidly degrade indoxacarb. It has a high degradation efficiency for indoxacarb, and the degradation rate of indoxacarb at a concentration of 10 mg / L by Rhodococcus pyridinivorans DPX-f2 reached 77% in a basal salt medium with indoxacarb as the sole carbon source.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pyridine-eating Rhodococcus faecalis that degrades indoxacarb, characterized in that: The Rhodococcus pyridinivorans strain is classified and named Rhodococcus pyridinivorans. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 14, 2026, with accession number CGMCC NO: 37394.

2. The application of Rhodococcus pyridostigma as described in claim 1 in the degradation of indoxacarb.

3. The application according to claim 2, characterized in that: The *Rhodococcus pyridae* species can degrade indoxacarb at concentrations below 100 mg / L within a pH range of 5-9 and a temperature range of 20-30°C.

4. The application according to claim 2, characterized in that: The *Rhodococcus pyridae* strain achieved a 77% degradation rate of 10 mg / L indoxacarb in a basal salt medium with indoxacarb as the sole carbon source.

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