Sinorhizobium meliloti and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses sinorhizobium meliloti and application thereof. The invention provides a strain of sinorhizobium meliloti, the classification name of the sinorhizobium meliloti is sinorhizobium meliloti, the sinorhizobium meliloti is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.36886. The sinorhizobium medicaginis provided by the invention can efficiently degrade abamectin, the abamectin degradation rate of the sinorhizobium medicaginis provided by the invention is obviously higher than that of various reported degradation strains, the sinorhizobium medicaginis shows excellent degradation efficiency and environmental adaptability, and the sinorhizobium medicaginis can effectively degrade abamectin in soil and can realize degradation of abamectin in grape fruits.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and discloses a strain of *Rhizobium sinense* from alfalfa and its applications. Background Technology

[0002] Avermectin (AVM) is a lipid-soluble macrolide insecticide, stable at room temperature, readily soluble in organic solvents but poorly soluble in water. Its excellent insecticidal activity has led to its widespread use in global agriculture, but its high lipid solubility and difficulty in natural degradation result in persistent residues and accumulation in the environment. These residues not only induce microbial resistance but also pose potential threats to ecosystems and human health. Currently, various technologies have been developed for the removal of avermectin residues, mainly including photocatalytic degradation, thermal decomposition, and microbial degradation. Among these, microbial degradation has attracted much attention due to its low cost and environmentally friendly characteristics. Currently reported functional microorganisms involve multiple genera; for example, strains of Bacillus spp. and Burkholderia sp. have been shown to directly degrade avermectin using it as a carbon source. However, these natural strains generally face bottlenecks such as limited degradation efficiency, weak environmental adaptability, and insufficient application stability. These bottlenecks have been widely observed and reported in the field and fruit applications of pesticide-degrading microbial agents, severely restricting their practical large-scale application. Therefore, identifying natural degrading bacteria with strong environmental adaptability, high degradation capacity, and wide applicability is crucial for developing highly effective biological agents. Summary of the Invention

[0003] To overcome the aforementioned problems in the prior art, this invention provides a strain of *Rhizobium sinense* from alfalfa and its applications. Compared to the prior art, the advantage of this invention lies in providing for the first time a strain of *Rhizobium sinense* from alfalfa, TLF6-3, with highly efficient avermectin degradation capabilities. This strain not only possesses the inherent plant symbiotic characteristics and environmental adaptability of rhizobia, but also exhibits significant degradation function for avermectin, broadening the resource range of pesticide-degrading bacteria. This strain has high degradation efficiency; under experimental conditions, its degradation rate of avermectin is significantly better than most reported degrading bacteria, demonstrating excellent pesticide decomposition potential. Strain TLF6-3 grows and reproduces rapidly, exhibits strong resistance, and can maintain degradation activity under various environmental conditions, possessing good potential for field application. This strain is non-toxic and harmless, and can be used directly or in formulations for the degradation of avermectin residues in agricultural soils, water bodies, and fruits, possessing the dual functions of increasing agricultural yield and quality while protecting the environment.

[0004] In the technical solution provided by this invention, "TLF6-3", "strain TLF6-3" or "Alfalfa Rhizobium TLF6-3" and "Avermectin Degrading Bacterium" all refer to the alfalfa rhizobium provided by this invention.

[0005] On one hand, this invention relates to a strain of *Rhizobium sinense* from alfalfa, the preservation information of which is as follows:

[0006] Strain name: *Alfalfa rhizobium* TLF6-3; Classification and nomenclature: *Alfalfa rhizobium* Sinorhizobium meliloti ; Preservation period: December 4, 2025; Preservation institution: China General Microbiological Culture Collection Center; Accession number: CGMCC No. 36886; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] On the other hand, the present invention relates to the application of the aforementioned *Alfalfa rhizobium* in the degradation of avermectin.

[0008] Furthermore, in the application provided by the present invention, the pH of the degradation environment is 6.0~8.0, the temperature is 25~40℃, and the concentration of abamectin is 20~100mg / L.

[0009] Furthermore, in the application provided by the present invention, the degradation environment has a pH of 7.0, a temperature of 35°C, and an abamectin concentration of 60 mg / L.

[0010] On the other hand, the present invention relates to a microbial inoculant, the active ingredients of which include the supernatant, fermentation product, filtrate or extract of the aforementioned *Rhizobium sinense* and / or its culture.

[0011] On the other hand, the present invention relates to a formulation for degrading avermectin in the environment, the active ingredient of which includes the supernatant, fermentation product, filtrate or extract of the aforementioned *Rhizobium sinense* and / or its culture.

[0012] Any of the *Rhizobium sinense* or any active variant thereof provided herein, such as combinations of hyphae, can be formulated into mycelial pastes, wettable powders, mycelial clumps, dust, granules, slurries, dry powders, aqueous or oil-based liquid products, etc. Such formulations will contain *Rhizobium sinense* or its active variants and / or compositions derived therefrom provided herein, along with carriers and other reagents. The formulations can be used in a variety of methods disclosed elsewhere herein.

[0013] The various compositions and formulations disclosed herein may contain a certain amount of hyphae of *Sinobacterium spp.* alfalfa or its active variants; and / or may contain a certain amount of composition derived from any of *Sinobacterium spp.* alfalfa or any of its active variants. Such amounts may include at least about 10 4 CFU / mL to approximately 10 11 CFU / mL, at least approximately 105 CFU / mL to approximately 10 11 CFU / mL, approximately 10 5 CFU / mL to approximately 10 10 CFU / mL, approximately 10 5 CFU / mL to approximately 10 12 CFU / mL, approximately 10 5 CFU / mL to approximately 10 6 CFU / mL, approximately 10 6 CFU / mL to approximately 10 7 CFU / mL, approximately 10 7 CFU / mL to approximately 10 8 CFU / mL, approximately 10 8 CFU / mL to approximately 10 9 CFU / mL, approximately 10 9 CFU / mL to approximately 10 10 CFU / mL, approximately 10 10 CFU / mL to approximately 10 11 CFU / mL or approximately 10 11 CFU / mL to approximately 10 12 The strain concentration is CFU / mL. In other embodiments, the strain concentration includes at least approximately 10. 4 CFU / mL, at least approximately 10 5 CFU / mL, at least approximately 10 6 CFU / mL, at least approximately 10 7 CFU / mL, at least approximately 10 8 CFU / mL, at least approximately 10 9 CFU / mL, at least approximately 10 10 CFU / mL, at least approximately 10 11 CFU / mL, at least approximately 10 12 CFU / mL. The strain at the above concentration can be formed in any type of formulation for any purpose, including, for example, in liquid formulations, wettable powders, sprayed dry formulations, mycelial pastes, wettable granules, or freeze-dried formulations.

[0014] As a preferred embodiment of the microbial agent, the agent is applied after being processed into an agriculturally acceptable formulation. Those skilled in the art will understand that the microbial agent provided by this invention is typically used in agriculture in the form of formulations (such as wettable powders, suspensions, pastes, etc.), containing not only the active ingredient (supernatant, fermentation product, filtrate, or extract of *Rhizobium sinense* and / or the culture of *Rhizobium sinense*), but also an agriculturally acceptable carrier or adjuvant.

[0015] For example, the active ingredient (agaricus rhizobium and / or the supernatant, fermentation product, filtrate or extract of the culture of agaricus rhizobium) can be prepared as a conventional wettable powder or granule or dispersible granule, which is well known to those skilled in the art using appropriate carriers or adjuvants. Suitable dispersants include polycarboxylates (TERSPERSE2700, T36, GY-D06, etc.), lignin sulfonates (Ufoxane 3A, Borresperse NA, Borresperse CA-SA, etc.), naphthalene and alkylnaphthalene formaldehyde condensate sulfonates (NNO, MF, Morwet D-425, Tamol NN, TERSPERSE2020, etc.), dispersant BX (sodium dibutylnaphthalene sulfonate), EO-PO block polyethers, alkylphenol polyoxyethylene ether phosphates, and alkylphenol polyoxyethylene ether formaldehyde condensate sulfates (SOPA), one or more of the following; wetting agents include sulfates (K-12), sulfonates (ABS-Na, BX, Terwet 1004, etc.), and composite wetting agents (Morwet...). One or more of the following: EFW; fillers such as diatomaceous earth, kaolin, light calcium carbonate, talc, silica, attapulgite, clay, ammonium sulfate, urea, sucrose, glucose, corn starch, sodium sulfate, sodium polyphosphate, etc.

[0016] For example, the active ingredient (supernatant, fermentation product, filtrate, or extract of *Rhizobium sinense* and / or culture of *Rhizobium sinense*) can be prepared as a conventional suspension. Dispersants that can be used to prepare the suspension include one or more of polycarboxylates, lignin sulfonates, alkyl naphthalene sulfonates (dispersant NNO), and TERSPERSE 2020 (produced by Huntsman, Inc., USA, an alkyl naphthalene sulfonate); emulsifiers such as the BY (castor oil polyoxyethylene ether) series emulsifiers (BY-110, BY-125, BY-140), Agricultural Emulsifier 700# (generic name: alkylphenol formaldehyde resin polyoxyethylene ether), Agricultural Emulsifier 2201, Span-60# (generic name: sorbitan monostearate), Tween-60# (generic name: dehydrated sorbitan monostearate polyoxyethylene ether), Agricultural Emulsifier 1601# (generic name: phenethylphenol polyoxyethylene polyoxypropylene ether), and TERSPERSE... One or more of 4894 (produced by Huntsman Corporation, USA); wetting agents such as alkylphenol polyoxyethylene ether formaldehyde condensate sulfate, alkylphenol polyoxyethylene ether phosphate, phenethylphenol polyoxyethylene ether phosphate, alkyl sulfate, alkyl sulfonate, naphthalene sulfonate, TERSPERSE 2500 (produced by Huntsman Corporation, USA) or one or more; thickeners such as silica, polyvinyl alcohol, bentonite, magnesium aluminum silicate or one or more; safety agent MON13900; antifreeze agents such as ethylene glycol, propylene glycol, glycerin, urea, inorganic salts such as sodium chloride or one or more; dispersion media such as soybean oil, rapeseed oil, cottonseed oil, corn oil, castor oil, palm oil, epoxidized soybean oil, methyl oleate and its methylated oils, diesel oil, engine oil, mineral oil, and ester solvents such as dimethyl phthalate, dibutyl phthalate, ethyl acetate, methyl benzoate or one or more.

[0017] Exemplarily, the active ingredient (*Rhizobium sinense* and / or the supernatant, fermentation product, filtrate, or extract of a culture of *Rhizobium sinense*) can be prepared into conventional granules. Those skilled in the art can select a core material made of diatomaceous earth, kaolin, light calcium carbonate, talc, silica, attapulgite, clay, etc., and use disc granulation technology to adhere (adsorb) the active ingredient onto the surface of the core material. In another preferred embodiment, those skilled in the art can prepare the granules into core-shell particles with a multi-layered structure, where each layer may contain the same or different components.

[0018] On the other hand, the present invention relates to a method for reducing the concentration of avermectin in the environment, comprising: causing the alfalfa rhizobium to act on an environment containing avermectin.

[0019] Furthermore, in the method for reducing the concentration of avermectin in the environment provided by the present invention, the environment is soil, water, or fruit.

[0020] Furthermore, in the method for reducing the concentration of avermectin in the environment provided by the present invention, the environment containing avermectin has a pH of 6.0 to 8.0, a temperature of 25 to 40°C, and an avermectin concentration of 20 to 100 mg / L.

[0021] Furthermore, in the method for reducing the concentration of avermectin in the environment provided by the present invention, the environment containing avermectin has a pH of 7.0, a temperature of 35°C, and an avermectin concentration of 60 mg / L.

[0022] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: This invention successfully isolated *Sinobacterium spp.* TLF6-3 from soil and leaf samples of a vineyard in Turpan that had been contaminated with avermectin for a long period. Morphological, physiological, biochemical, and 16S rDNA sequence analyses confirmed its taxonomic position as *Sinobacterium spp.* (Alfalfa rhizobia). Sinorhizobium meliloti Systematic analysis of its degradation characteristics revealed that under conditions of pH 7.0, 35℃, substrate concentration of 60 mg / L, and inoculum size of 2%, the degradation rate of this strain reached a maximum of 68.54% after 7 days, significantly higher than that of several previously reported degrading strains, demonstrating excellent degradation efficiency and environmental adaptability. TLF6-3 maintained high activity across a wide environmental range of pH 6.0–8.0 and temperature 25–40℃, indicating good ecological adaptability and application stability. Appropriate substrate concentrations effectively induced degradation metabolic pathways, while excessively high concentrations showed a slight inhibitory effect. Inoculum size variation experiments further demonstrated that population density and metabolic balance jointly influenced the degradation rate, reflecting the strain's dynamic regulatory ability in response to external nutrient and stress signals. Genome-wide functional annotation results showed that the avermectin-degrading bacterium TLF6-3 was enriched with multiple functional genes related to transmembrane transport, multi-enzyme catalysis, energy metabolism, and stress response. Based on existing literature reports, it is speculated that these genes may synergistically participate in the degradation process of avermectin, providing a potential molecular mechanism for its transformation within the strain. In summary, this study provides a highly efficient microbial strain resource for the bioremediation of avermectin-contaminated soil or the degradation of contaminated fruits, and elucidates the molecular basis of TLF6-3 degradation function at the genomic level, providing theoretical support for subsequent verification of key enzyme genes and development of microbial remediation agents. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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.

[0024] Figure 1 The image shows the screening results of avermectin-degrading strains. A represents strain TLF2-6; B represents strain TLF3-2; C represents strain TLF4-1; D represents strain TLF4-4; and E represents strain TLF6-3.

[0025] Figure 2 The degradation rates of different TLF strains are shown. Data are expressed as mean ± standard deviation (SD); different lowercase letters in the bar chart indicate statistically significant differences between groups. p <0.05).

[0026] Figure 3 The images show the colony morphology of strain TLF6-3. A shows the colony morphology of TLF6-3 on YMA solid medium; B shows the Gram-stained optical microscopic observation of TLF6-3 (×100).

[0027] Figure 4 This is a phylogenetic tree diagram of strain TLF6-3.

[0028] Figure 5 Distribution diagram of COG functional annotation for strain TLF6-3.

[0029] Figure 6 Distribution of GO functional annotations for strain TLF6-3.

[0030] Figure 7 Distribution diagram of KEGG functional annotation for strain TLF6-3.

[0031] Figure 8 This is a circular diagram of the genome of strain TLF6-3. Detailed Implementation

[0032] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially. Unless otherwise specified, % and ‰ in the following embodiments refer to mass percentage and mass thousandths of content.

[0033] Example 1 This embodiment provides a screening method for avermectin-degrading strains.

[0034] Soil and leaf samples were collected from vineyards in Grape Town, Turpan, where abamectin had been applied for a long period of time.

[0035] Basic salt medium (g / L): NaCl 1.00 g, (NH4)2SO4 1.00 g, K2HPO4 1.50 g, KH2PO4 0.50 g, MgSO4 0.20 g, pH 7.0~7.5; enrichment medium (avermectin medium) is basic salt medium with avermectin concentration added to 100 mg / L.

[0036] Using scissors, cut the stems and leaves (leaf age 2 months) of seedless white grapes harvested in Turpan into uniform shapes. Wipe the grape leaves with 75% anhydrous ethanol, let them dry, add 5 mL of sterile water, extract the juice using a juicer, filter it through four layers of gauze, and take 1 mL of the supernatant from the filter into a 1.5 mL centrifuge tube. Use a serial dilution method to dilute to 10. -2 10 -3 10 -4 and 10 -5 The bacteria were evenly spread on NA medium using a spreader and incubated at 30°C for 7 days. Single colonies were picked according to their morphology and streaked onto NA medium for purification.

[0037] Ten soil samples (5g each) were weighed and added to 100mL of sterile water. The samples were incubated at 30℃ and 140rpm for 24 hours with shaking. After incubation, the samples were allowed to stand for 1 hour, and the supernatant was transferred to 1.5mL centrifuge tubes. The samples were then diluted to 10⁻⁶ using a serial dilution method. -2 10 -3 10 -4 and 10 -5 The bacteria were evenly spread on NA and culture medium using a spreading stick and incubated at 30°C for 7 days. Single colonies were picked according to their morphology and streaked onto NA medium for purification.

[0038] A culture medium with avermectin as the sole carbon source was prepared. Microorganisms isolated and purified from soil and leaves were inoculated onto this culture medium. After the colonies grew to be visible to the naked eye, avermectin was extracted and the recovery rate was determined.

[0039] Once the enrichment medium was confirmed to have a degradation effect, it was serially diluted and evenly spread on 1 / 10 LB agar plates containing 60 mg / L avermectin, and then incubated at 30°C. After single colonies grew on the plates, these single colonies were selected and transferred to basal salt medium containing 60 mg / L avermectin to verify the degradation ability of these single bacteria on avermectin.

[0040] Microorganisms isolated and purified from soil and leaves were inoculated onto a culture medium using avermectin as the sole carbon source. After the colonies grew to be visible to the naked eye, avermectin extraction and degradation recovery rates were determined. The selected strains were inoculated onto avermectin (100 mg / L) inorganic salt medium, with initial bacterial concentrations (OD)...600 =0.2, with uninoculated avermectin medium as a control, and cultured at 37℃ for 7 days. After culture, the avermectin content was determined using a Waters Acquity UPLC tandem quadrupole (TQD) mass spectrometer. The Waters Acquity UPLC tandem quadrupole (TQD) mass spectrometer used an Acquity UPLC HSS C column. 18 (1.7 μm, 2.1 × 100 mm). Column temperature was 40 °C, mobile phase was methanol-pure water (93:7, v / v), flow rate was 0.3 min / mL, and injection volume was 2 μL. MS / MS analysis was performed in negative ion mode with capillary voltage 2.5 kV, desolventizing temperature 600 °C, source temperature 125 °C, sheath gas flow rate 50 Arb, desolventizing gas flow rate 1000 L / h, and nitrogen flow rate 150 L / h.

[0041] Sample preparation: Pipette 10 mL of sample into a 50 mL plastic centrifuge tube, add 10 mL of acetonitrile and one ceramic homogenizer, and shake vigorously for 1 min. Add 4 g of anhydrous magnesium sulfate, 1 g of sodium chloride, 1 g of sodium citrate dihydrate, and 0.5 g of disodium citrate sesquihydrate, shake vigorously for 1 min, and centrifuge at 4200 rpm for 5 min. Quantitatively transfer the supernatant to a plastic centrifuge tube containing dehydrating agent and purification material (150 mg of anhydrous magnesium sulfate and 25 mg of PSA per ml of extract); vortex to mix for 1 min. Centrifuge at 4200 rpm for 5 min, and filter the supernatant through a microporous membrane for analysis.

[0042] First, perform a primary stain with ammonium oxalate crystal violet for 1 minute, then rinse with running water. Next, mordate with Gram's iodine solution for 1 minute, then rinse with running water again. Then, destain with 95% alcohol until no more purple smears off, rinsing again with running water after approximately 20-30 seconds. Finally, counterstain with safranin for 1 minute, rinse, and allow to air dry. Observe the slide after it is completely dry.

[0043] Based on the "Handbook for Systematic Identification of Common Bacteria", the isolated endophytic bacteria were subjected to physiological and biochemical tests using the API20E kit.

[0044] Following existing literature, bacterial genomic extraction kits were used to extract bacterial DNA. Total DNA was used as a template, and PCR was performed using universal primers 27F and 1492R. Amplification was performed according to existing literature, using a 30 μL amplification system: 2 μL template DNA, 1 μL each primer, 15 μL 2×PCR Buffer Mix, and 11 μL ddH2O. The amplification program was: 94℃ for 4 min; 94℃ for 30 s, 65℃ for 40 s, 72℃ for 90 s, 30 cycles; 72℃ for 10 min. After passing 1% agarose gel electrophoresis, the amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequenced sequences were submitted to the GenBank database. Comparative analysis with NCBI and EzBioCloud databases was performed, and 16S rDNA sequences from similar strains were downloaded. A phylogenetic tree was constructed using MEGA 11.0 software and the NJ method, with a Bootstrap value of 1000.

[0045] After the amplified products were confirmed to be acceptable by 1% agarose gel electrophoresis, they were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Following sequencing, the obtained sequences were submitted to the GenBank database and compared and analyzed on the NCBI and EzBioCloud platforms. Based on the sequencing results, a phylogenetic tree was constructed using Mega11.0 software.

[0046] All strains were screened for avermectin resistance and cultured on LB solid medium at 30°C before colony morphology was observed. Figure 1 As shown, by enriching and culturing bacteria in soil and leaf samples, a total of 5 strains resistant to avermectin were isolated, namely TLF2-6, TLF3-2, TLF4-1, TLF4-4, and TLF6-3.

[0047] like Figure 2 The average degradation rates of strains TLF2-6, TLF3-4, TLF4-4, TLF4-8, and TLF6-3 obtained from the initial screening were 33.97%, 21.40%, 37.45%, 40.62%, and 49.39%, respectively. Based on the degradation ability, the strain with the strongest degradation ability was finally selected as the subject of subsequent research, namely TLF6-3.

[0048] Morphological observation and Gram staining of TLF6-3 were performed, and the results are shown in the figure. Figure 3 After culturing on YMA solid medium at 28℃ for 3-5 days, TLF6-3 colonies were milky white, round, raised, smooth, and moist, with a diameter of approximately 1.0-2.0 mm, neat edges, and a semi-transparent appearance. Microscopic observation revealed that this strain was a Gram-negative bacterium, with rod-shaped cells, blunt ends, and no spores. Based on this, it was preliminarily identified as belonging to the Rhizobium family (Rhizobium). Rhizobiaceae Related strains.

[0049] The physiological and biochemical characteristics of strain TLF6-3 are shown in Table 1. This strain was positive for β-galactosidase, arginine dihydrolase, and ornithine decarboxylase tests. It cannot utilize citrate as its sole carbon source and does not ferment glucose, mannitol, or sorbitol to produce acid. However, it was negative for lysine decarboxylase, hydrogen sulfide production, urease, and tryptophan deaminase tests, and it did not produce indole. A positive Voges-Proskauer (VP) test indicates that it can produce acetylmethane. Furthermore, this strain cannot liquefy gelatin and cannot ferment inositol and rhamnose. These physiological and biochemical characteristics are consistent with those of *Rhizobium sinense* (a genus of bacteria). Sinorhizobium This aligns with the typical characteristics of ( ).

[0050] Table 1: Physiological and biochemical identification results of strain TLF6-3

[0051] The 16S rDNA sequencing results of strain TLF6-3 were compared with those in the NCBI database. The results showed that this bacterium was similar to *Sinium spp.* in the database. Sinorhizobium meliloti The similarity to *Alfalfa sinensis* reached over 97%, therefore it was identified as *Alfalfa sinensis* (…). Sinorhizobium meliloti The standard strains from the BLAST results were selected, and a phylogenetic tree was constructed using the biological software MEGA 11.0. See [link to results]. Figure 4 . Figure 4 The values ​​at the middle branch nodes represent the confidence level of 1000 bootstrap replicate tests (only confidence levels ≥50% are displayed); the scale bar represents a nucleotide sequence difference of 0.05; the strain isolated and identified in this study is TLF6-3; the sequences of other strains are all from the NCBI database (strain numbers are labeled after the species name).

[0052] Example 2 This embodiment demonstrates the degradation performance of TLF6-3 on avermectin.

[0053] (1) Effect of pH on the degradation growth and degradation rate of avermectin-degrading bacteria.

[0054] To investigate the growth and degradation capacity of avermectin-degrading bacteria under different pH conditions, the culture results of the strains in the pH range of 5.0 to 9.0 were analyzed (Table 2).

[0055] Table 2: Effect of pH on the growth and degradation rate of avermectin-degrading bacteria

[0056] Note: Data are expressed as mean ± standard deviation. Different lowercase letters in the same row indicate that the results were obtained after one-way ANOVA and Tukey's HSD post-hoc test. p There were significant differences at the <0.05 level.

[0057] The strain achieved its highest growth rate, reaching 8.39 log₂O₅, at an initial pH of 7.0. 10 (CFU / mL), and the degradation rate of avermectin also reached its maximum (45.19%), significantly higher than other pH treatment groups. p <0.05). Under pH 6.0 and pH 8.0 conditions, the strains exhibited good growth ability, with degradation rates of 28.14% and 43.23%, respectively, which were significantly different from the optimal pH condition (pH 7.0). p <0.05). When the initial pH was lowered to 5.0 or raised to 9.0, the growth rate and avermectin degradation rate of the strain both decreased significantly, with degradation rates of 13.43% and 39.83%, respectively. In summary, the avermectin-degrading bacteria grew most vigorously and had the highest degradation efficiency under neutral conditions, indicating that pH 7.0 was the optimal growth and degradation condition.

[0058] (2) Effect of temperature on the growth and degradation rate of avermectin-degrading bacteria.

[0059] To investigate the effect of temperature on the growth and degradation capacity of avermectin-degrading bacteria, the strains were cultured at 20℃, 25℃, 30℃, 35℃ and 40℃, and the bacterial growth and avermectin degradation rate were measured (Table 3).

[0060] Table 3: Effects of temperature on the growth and degradation rate of avermectin-degrading bacteria

[0061] Note: Data are expressed as mean ± standard deviation. Different lowercase letters in the same row indicate that the results were obtained after one-way ANOVA and Tukey's HSD post-hoc test. p There were significant differences at the <0.05 level.

[0062] The results showed that temperature affected both the growth and degradation of the strain. At low temperatures of 20℃ and 25℃, the degradation rates were 21.58% and 29.31%, respectively. As the temperature increased to 30℃ and 35℃, the degradation rate continued to increase, reaching its highest value of 52.72% at 35℃, approximately 1.4 times higher than at 20℃. However, when the temperature continued to rise to 40℃, the degradation rate decreased to 48.58%, lower than the optimum temperature but higher than at low temperatures. The growth rate followed the same trend as the degradation rate, gradually increasing with temperature, peaking at 35℃, and then slightly decreasing.

[0063] In conclusion, the avermectin-degrading bacteria exhibit the strongest growth activity and degradation capacity at 35℃, therefore, the optimal culture temperature is determined to be 35℃.

[0064] (3) Effect of substrate concentration on the growth and degradation rate of avermectin-degrading bacteria.

[0065] To investigate the effects of different substrate concentrations on the growth and degradation efficiency of avermectin-degrading bacteria, avermectin concentrations of 20, 40, 60, 80, and 100 mg / L were set in inorganic salt medium, and the strains were cultured and their growth and degradation rates were measured (Table 4).

[0066] Table 4: Effect of substrate concentration on the growth and degradation rate of avermectin-degrading bacteria

[0067] Note: Data are expressed as mean ± standard deviation. Different lowercase letters in the same row indicate that the results were obtained after one-way ANOVA and Tukey's HSD post-hoc test. p There were significant differences at the <0.05 level.

[0068] The results showed that the degradation rate of TLF6-3 under different substrate concentrations exhibited a concentration-dependent change. p <0.05).

[0069] At low concentrations of 20 mg / L and 40 mg / L, the degradation rates were 29.52% and 43.43%, respectively. As the concentration increased to 60 mg / L, the degradation rate further increased to 65.64%, the highest among all concentrations, more than 1.2 times higher than at 20 mg / L. When the substrate concentration continued to increase to 80 mg / L and 100 mg / L, the degradation rates decreased to 61.49% and 50.38%, respectively, showing a trend of "optimal at medium concentrations followed by a decrease at higher concentrations."

[0070] Therefore, an appropriate substrate concentration can promote the growth and degradation reaction of avermectin-degrading bacteria, while excessively high substrate concentrations may inhibit bacterial cell growth. The overall results indicate that 60 mg / L is the optimal substrate concentration for this strain to degrade avermectin.

[0071] (4) Effect of inoculum amount on the growth and degradation rate of avermectin-degrading bacteria.

[0072] To investigate the effect of different inoculum amounts on the degradation effect of avermectin-degrading bacteria, five inoculum ratios of 1%, 1.5%, 2%, 2.5%, and 3% were set under the same culture conditions, and the avermectin degradation rate of each treatment group was measured (Table 5).

[0073] Table 5: Effect of inoculum size on the growth and degradation rate of avermectin-degrading bacteria

[0074] Note: Data are expressed as mean ± standard deviation. Different lowercase letters in the same row indicate that the results were obtained after one-way ANOVA and Tukey's HSD post-hoc test. p There were significant differences at the <0.05 level.

[0075] The results showed that the inoculum size had a significant effect on the degradation ability of the strain. p <0.05).

[0076] When the inoculum concentration was 1% and 1.5%, the degradation rates were 27.55% and 44.58%, respectively. As the inoculum concentration increased to 2%, the degradation rate reached its highest value of 68.54%, more than 1.5 times higher than the 1% condition. Subsequently, when the inoculum concentration was further increased to 2.5% and 3%, the degradation rates were 65.13% and 64.08%, respectively, showing a slight downward trend. An appropriate inoculum concentration is beneficial for the bacteria to quickly adapt to the environmental pressure of avermectin, thereby improving degradation efficiency; however, excessively high inoculum concentrations may lead to a relative deficiency of nutrients such as carbon sources, resulting in increased competition for bacterial growth and thus reducing degradation activity. The overall results indicate that 2% is the optimal inoculum concentration for this strain to degrade avermectin.

[0077] Example 3 This example provides a genomic analysis of TLF6-3.

[0078] After obtaining the gene set of the sequenced strain, the genes need to be compared and annotated with databases to determine their functions and related descriptive information. This process can reflect the overall functional classification of the strain's gene set and facilitate subsequent research to identify target functional genes.

[0079] The COG database can be divided into twenty-five categories according to function, and the statistical results are as follows: Figure 5 As shown. Figure 5 The results showed that in the metabolism category, genes related to "amino acid transport and metabolism" (595 genes) and "carbohydrate transport and metabolism" (573 genes) were highly enriched, providing a genetic basis for the metabolism of matter and energy for avermectin degradation; "secondary metabolite biosynthesis, transport and catabolism" (97 genes) directly pointed to the degradation pathway of avermectin (a polyketide secondary metabolite xenobiotic), highlighting its metabolic potential.

[0080] GO stands for Gene Ontology, which is divided into three main categories: 1) Cellular Components: used to describe subcellular structures, locations, and macromolecular complexes, such as the nucleolus, telomeres, and recognition initiation complexes; 2) Molecular Functions: used to describe the individual functions of genes and gene products, such as carbohydrate binding or ATP hydrolase activity; 3) Biological Processes: used to describe the orderly combination of molecular functions to achieve broader biological functions, such as mitosis or purine metabolism. Genes are assigned to one or more categories based on the properties of their products. Through GO database annotation, we can determine the possible functions of genes based on their annotation in different categories. The statistical results of the three categories in the GO database for each sample are as follows: Figure 6 As shown. Figure 6 The results showed that, in biological processes, genes related to "cellular processes" (1964 genes) and "metabolic processes" (1568 genes) were enriched, providing a metabolic basis for avermectin degradation; in molecular functions, genes related to "catalytic activity" (2069 genes) and "binding activity" (1479 genes) were dominant, suggesting that they encode a large number of degradation-related enzymes; in cellular components, genes related to "cellular anatomy" (812 genes) were abundant, reflecting the support of cellular structure for degradation function.

[0081] KEGG database annotation allows for easy identification of all annotated genes associated with a specific function. The bar chart obtained after KEGG secondary classification statistics for each sample is shown below. Figure 7 As shown. Figure 7The results showed a significant enrichment of genes related to "transport and catabolic metabolism" (395 genes) and "membrane transport" (220 genes) in cellular processes. The former provides key metabolic support for the intracellular degradation of avermectin, while the latter, by encoding elements such as ABC transporters and oligopeptide transporters, ensures the substrate's entry into intracellular degradation sites. Multiple gene clusters related to transmembrane transport, redox reactions, and hydrolysis were predicted in TLF6-3, suggesting that these functional elements may synergistically participate in the molecular transformation process of avermectin, a "polyketide xenobiotic." Similar multi-enzyme synergistic patterns have been reported in studies on the degradation of xenobiotics in microorganisms, emphasizing the core roles of oxidoreductases and hydrolases in the degradation of complex organic matter. The "Global and Overview Maps" (1240 genes) provide systematic support at the overall metabolic network level, supplying energy and cofactors to the degradation process through core metabolic pathways such as glycolysis and the tricarboxylic acid cycle. From the four major pathways, core genes that are directly related to or key to the degradation of avermectin were further screened out. Their functional characteristics and association with degradation are shown in Table 6.

[0082] Table 6: Integration of core associated genes for avermectin degradation in TLF6-3 strain

[0083] Table Notes: 1) Data are derived from whole-genome sequencing results of TLF6-3 strain; 2) Screening criteria: core key genes (directly involved in key degradation steps: transmembrane uptake, structural disruption, cofactor supply), and important auxiliary genes (enhancing degradation efficiency, ensuring metabolic balance, and providing nutritional support) all meet the following criteria: E value < 1e-117 and sequence similarity > 87.5%; 3) All gene functions are directly related to or provide core support for avermectin degradation. The correlation and significance were verified by pathway enrichment analysis (…). p <0.05) and similar studies have verified this.

[0084] Combining pathway enrichment results with functional annotation screening, a total of 11 cytochrome P450 family genes were identified (Table 7). These genes are all associated with the core P450 metabolic pathways map00980 (drug metabolism) and map00982 (exogenous substance metabolism). The annotation reliability was rigorously verified. All genes had E values ​​<1e-117 (range 3.5e-117~1.9e-148) and sequence similarity >86%, which is far below the high reliability annotation threshold (E value <1e-5), confirming the accuracy of their functional annotation. In summary, the KEGG annotation results echo the high enrichment of genes related to "catalytic activity" in the GO functional classification (2069 genes) and the involvement of genes related to "secondary metabolite biosynthesis, transport, and catabolism" in the COG functional classification (97 genes). Through multi-dimensional data integration, it is inferred that the cytochrome P450 family genes, combined with the synergistic effects of pathways such as membrane transport and xenobiotic degradation, constitute the molecular basis for the degradation of avermectin by the TLF6-3 strain. These genes, with their broad-spectrum catalytic activity, are expected to drive the structural modification and degradation of avermectin through reactions such as oxidation and hydroxylation.

[0085] Table 7: Association between cytochrome P450 gene and avermectin degradation function in TLF6-3 strain

[0086] Table notes: 1) Data are derived from the whole genome sequencing results of TLF6-3 strain; 2) E values ​​are all <1e-117, and the sequence similarity is >86%, confirming the reliability of P450 gene annotation and providing a molecular basis for inferring degradation function; 3) The optimal degradation efficiency of the matched strain is 68.54% after 7 days when the degradation stage is divided.

[0087] By integrating the functions of core related genes, the molecular mechanism of avermectin degradation in strain TLF6-3 was elucidated: a transmembrane uptake transport system, primarily abcA and secondarily oppB, brings avermectin into the cell, while mdlB excretes the toxic product; structural disruption occurs through cyp11a1 breaking side chains, estA hydrolyzing ester bonds, and fmo / cyp21a2 oxidizing the benzene ring, achieving both avermectin activity disruption and structural simplification; metabolic support relies on ureC providing carbon and nitrogen sources and acdS eliminating stress toxins, ensuring continuous degradation; systemic support is provided by hexokinase / g6pd providing ATP and NADPH, maintaining the core enzyme activity and metabolic flux balance. The E values ​​of all related key genes are <1e-50, and the sequence similarity is >87.5%, indicating extremely high annotation reliability. Furthermore, the functions cover the entire avermectin degradation process, providing a clear molecular basis for the strain achieving a 68.54% degradation rate under suitable conditions (pH 7.0, 35℃, 60 mg / L substrate).

[0088] Based on the high enrichment of genes related to "catalytic activity" in the GO functional classification (2069 genes), the clear focus on the "xenobiotic biodegradation and metabolism" pathway in the KEGG pathway (151 genes), the involvement of genes related to "secondary metabolite biosynthesis, transport, and catabolism" in the COG functional classification (97 genes), and the visualization support of the physical distribution of functional genes by genome and plasmid loop maps, this invention, through multi-omics data integration and literature comparison, ultimately infers that the cytochrome P450 gene family may be the core degradation genes of the avermectin-degrading bacterium TLF6-3. This gene family, with its broad-spectrum catalytic activity, is expected to drive the structural modification and degradation of avermectin through reactions such as oxidation and hydroxylation.

[0089] Based on the analysis of the sequencing samples, Circos software was used to comprehensively display information such as genes, ncRNAs, repetitive sequences, annotation information, GC content, and GCskew on the genome of the sequenced strain. If the assembly reaches the level of a complete diagram, separate circos diagrams will be drawn for each genome sequence and plasmid sequence. Based on the sequenced bacterial genome sequence, GCskew analysis was performed using the (G–C) / (G+C) calculation method. Simultaneously, based on the gene distribution, ncRNA distribution, annotation, and other data results, the distribution of each element on the genome was displayed. The results are as follows: Figure 8 As shown. To analyze the genomic characteristics of the avermectin-degrading bacterium TLF6-3, Figure 8 This is a genomic loop diagram of TLF6-3. The genome size of this strain is 3,597,254 bp. The outermost layer of the loop diagram marks the physical locations of the genome (in MB), and the COG functional classification distribution of genes is displayed using color coding, visually presenting the physical layout of various functional genes on the genome. This provides a visual basis for exploring the regional clustering of genes related to avermectin degradation. The fluctuation of GC content in the middle loop (red peak) and GC skewness (green peak) reflect the base composition characteristics of the genome, which can be used to explore the potential rules of genome replication and transcriptional regulation. At the same time, the genomic localization of non-coding RNAs such as tRNA and rRNA reflects the genetic basis of gene expression and protein synthesis in this strain, providing support for its metabolic activity of avermectin degradation. This loop diagram provides genomic-level visual clues for elucidating the molecular mechanism of avermectin degradation in TLF6-3 from the dimensions of genomic physical structure, functional gene distribution, base composition, and non-coding RNA configuration.

[0090] Example 4 This embodiment provides a degradation function verification test of TLF6-3.

[0091] (1) Soil simulation degradation test of avermectin-degrading bacteria.

[0092] Weigh 20g of prepared test soil into a 250ml Erlenmeyer flask. Add avermectin at concentrations of 10mg / kg, 25mg / kg, and 50mg / kg, stir well with a glass rod, seal the flask, and add 100μL of avermectin-degrading bacterial fermentation broth to each soil sample. Perform the operation under aseptic conditions. A blank control group was set up for both groups. The treated soils were placed in an intelligent light incubator at 25℃, light intensity of 6500 lux, and a light-dark ratio of 12h:12h. Sterile water was added periodically during the experiment. Samples were collected on days D0, D1, D3, D7, D14, D28, D45, and D60. The concentration of avermectin in the soil was determined by high performance liquid chromatography-tandem mass spectrometry. Three parallel samples were prepared for each treatment. The experimental results are shown in Tables 8, 9, and 10. Data are expressed as mean ± standard deviation (n=3). Different lowercase letters in the same row indicate different values. p Significant differences exist at the <0.05 level, used to describe trends of change at different time points or under different treatment conditions.

[0093] Table 8: Changes in the degradation rate of abamectin in the soil simulation system (10 mg / L)

[0094] Table 9: Changes in the degradation rate of abamectin in the soil simulation system (25 mg / L)

[0095] Table 10: Changes in the degradation rate of abamectin in the soil simulation system (50 mg / L)

[0096] The results shown in Tables 8, 9, and 10 indicate that, under all three initial concentration conditions, the addition of bacteria significantly improved the degradation efficiency of abamectin in the soil simulation system. p <0.05). Under low concentration conditions (10 mg / L), the degradation rate of the bacterial treatment group increased significantly with the extension of culture time, reaching 91.62% at D60, which was significantly higher than the 78.40% of the CK group. Under medium concentration conditions (25 mg / L), the degradation rate of the bacterial treatment group reached 92.11% at the end of the culture period, which was also significantly higher than the CK group. When the initial concentration was increased to 50 mg / L, the degradation rate of avermectin decreased overall, but the bacterial treatment group still achieved a degradation rate of 72.45% at D60, which was significantly higher than the 50.63% of the CK group. This indicates that high concentration has a certain inhibitory effect on the degradation process, but the selected strains still have strong tolerance and continuous degradation ability.

[0097] (2) Simulated degradation test of grape fruit by avermectin-degrading bacteria.

[0098] Grapes with undamaged surfaces and uniform size, color, and ripeness were selected. Whole bunches of grapes were soaked in a fermentation liquid diluted 5 times; a control group was soaked in 0.2% sodium hypochlorite solution and then air-dried at room temperature for 2 hours. When no moisture remained on the surface of the grapes, they were stored at 25℃ for 7 days. Avermectin was extracted daily, and its degradation rate was analyzed. The experimental results are shown in Table 11. Data are expressed as mean ± standard deviation (n=3); different lowercase letters in the same row indicate... p Significant differences exist at the <0.05 level, used to describe trends of change at different time points or under different treatment conditions.

[0099] Table 11: Changes in the degradation rate of avermectin in a grape simulation system (25 mg / L)

[0100] Table 11 shows that the bacterial treatment significantly promoted the degradation process of abamectin in the grape simulation system. p <0.05). With prolonged culture time, the degradation rate of avermectin in the inoculum-treated group continuously increased, from 17.49% on D1 to 86.34% on D7, showing a clear time-dependent trend. In contrast, avermectin in the CK group mainly relied on natural decay, with a slower degradation rate; the degradation rate at the end of culture (D7) was only 28.44%, significantly lower than that of the inoculum-treated group. Furthermore, the standard deviation of the inoculum-treated group was smaller at each time point, indicating good experimental repeatability and that the strain possesses stable and efficient degradation capabilities in the grape simulation system. In conclusion, inoculum treatment can significantly improve the removal efficiency of avermectin in the grape simulation system, and the acute oral toxicity test of the strain showed it to be practically non-toxic, demonstrating the potential application value of this strain in agricultural product systems.

[0101] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A strain of *Rhizobium sinense* from alfalfa, characterized in that, The classification of *Sinium spp.* in alfalfa is named as follows: Sinorhizobium meliloti It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCCNo.36886.

2. The application of *Alfalfa Rhizobium sinense* as described in claim 1 in the degradation of avermectin.

3. The application according to claim 2, characterized in that, The degradation environment has a pH of 6.0~8.0, a temperature of 25~40℃, and an abamectin concentration of 20~100mg / L.

4. The application according to claim 3, characterized in that, The degradation environment had a pH of 7.0, a temperature of 35°C, and an abamectin concentration of 60 mg / L.

5. A microbial inoculant, characterized in that, The active ingredients include: the supernatant, fermentation product, filtrate, or extract of *Agropyron cristatum* and / or its culture as described in claim 1.

6. A formulation for degrading avermectin in the environment, characterized in that, The active ingredients include: the supernatant, fermentation product, filtrate or extract of *Alfalfa rhizobium sinense* and / or its culture as described in claim 1.

7. A method for reducing the concentration of avermectin in the environment, characterized in that, include: This allows the *Alfalfa Rhizobium sinense* of claim 1 to act on an environment containing abamectin.

8. The method for reducing the concentration of avermectin in the environment according to claim 7, characterized in that, The environment is soil, water, or fruit.

9. The method for reducing the concentration of avermectin in the environment according to claim 7, characterized in that, In the environment containing avermectin, the pH is 6.0~8.0, the temperature is 25~40℃, and the avermectin concentration is 20~100mg / L.

10. The method for reducing the concentration of avermectin in the environment according to claim 9, characterized in that, The environment containing abamectin was soil with a pH of 7.0, a temperature of 35°C, and an abamectin concentration of 60 mg / L.

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