Pseudomonas nitroreducens MHY01 and application thereof as thifluzamide synergist

Abstract

The invention discloses a pseudomonas nitroreducens MHY01 and an application of the pseudomonas nitroreducens MHY01 as a thifluzamide synergist. According to the invention, a strain capable of promoting rice to absorb thifluzamide is separated from healthy rice root soil, the strain is named as pseudomonas nitroreducens MHY01, and a composite flora capable of promoting pesticide absorption is further obtained. The strain and the composite flora can significantly reduce pesticide soil adsorption loss, reduce soil and water pollution risks, alleviate drug resistance development and promote agricultural sustainable development on the premise of ensuring the disease and pest control effect in practical application. And secondly, the strain is derived from healthy rice rhizosphere, is green and safe, has no obvious influence on rice growth, and ensures the quality safety and ecological safety of agricultural products. Therefore, the utilization efficiency of the pesticide is improved by developing rhizosphere microorganisms, and the problems that the utilization rate is low and the dosage is increased due to the fact that the pesticide is adsorbed by soil are fundamentally solved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to a strain of nitro-reducing Pseudomonas MHY01 and its application as a thifluzamide synergist. Background Technology

[0002] Pesticides are crucial for ensuring agricultural production, but the long-term excessive, frequent, and irrational use of pesticides in my country not only increases manufacturing costs but also leaves residues in the soil and water environment, damages soil ecology, threatens human health (through crop absorption and accumulation), and induces pesticide resistance in pests. For example, applying effective seed dressing agents (such as thifluzamide) to the roots can effectively control rice sheath blight (a single application can be effective for a long time), but some pesticides are adsorbed by the soil and not absorbed by crops, resulting in low utilization rates, increased dosage, and exacerbated farmland pollution. Therefore, reducing pesticide use and improving effective utilization rates are key challenges for achieving green agricultural development and ensuring the quality of agricultural products and the safety of the ecological environment.

[0003] Current strategies for improving pesticide utilization mainly include: (1) Improve pesticide formulation: Promote pesticide slow-release technology, and achieve fixed-point, quantitative and timed release of active ingredients by encapsulating them in materials, thereby reducing pesticide damage and losses and improving efficiency.

[0004] (2) Optimize plant protection equipment and application techniques: such as applying ultra-micro atomization technology (greenhouse) and electrostatic sprayers (to improve the uniformity of deposition), and select the best equipment combination according to the crop.

[0005] (3) Use pesticide adjuvants to improve the physical and chemical properties of the pesticide solution (such as surface tension, viscosity, droplet size).

[0006] The core of these methods is to increase the deposition of pesticides on targets (such as leaves). However, they generally overlook the crucial regulatory role of rhizosphere soil microorganisms in crop pesticide absorption. The crop rhizosphere possesses an extremely rich microbial community, among which certain rhizosphere bacteria can significantly influence the crop's absorption and utilization of external substances (including pesticides). Existing methods for improving pesticide utilization efficiency often neglect the impact of rhizosphere soil microorganisms on crop pesticide absorption, and no rhizosphere microorganisms capable of significantly influencing crop pesticide absorption and utilization have been developed. Therefore, based on the regulatory role of rhizosphere bacteria, developing microbial agents that promote pesticide absorption has significant value and broad prospects for significantly improving pesticide utilization, reducing dosage, and minimizing environmental pollution.

[0007] Patent CN111893061A discloses that *Pseudomonas nitroreductoids* CW-7 can be used to remediate pyrethroid insecticide-contaminated water bodies, soil, and other natural environments. Direct application for 7 days can reduce pyrethroid insecticide residues in water and soil by more than 95%. However, there are currently no reports on *Pseudomonas nitroreductoids* promoting the absorption and utilization of systemic pesticides by crops or reducing pesticide residues in the natural environment. Summary of the Invention

[0008] The purpose of this invention is to overcome the defects and shortcomings of existing technologies in agricultural production, such as large amounts of pesticides used and low effective utilization rates, resulting in serious farmland pollution, and to provide a rhizosphere microorganism, *Pseudomonas nitroreductoides* (…). Pseudomonas (sp.)MHY01, which promotes the absorption of pesticides by crops, increases the content of pesticides in crops, and improves the utilization rate of pesticides.

[0009] The second objective of this invention is to provide a complex microbial community.

[0010] A third objective of this invention is to provide a microbial agent.

[0011] The fourth objective of this invention is to provide the application of the above-mentioned nitro-reducing Pseudomonas MHY01, the above-mentioned complex microorganisms, or the above-mentioned microbial agents as thifluzamide synergists.

[0012] The fifth objective of this invention is to provide a method for promoting the absorption of thifluzamide in rice.

[0013] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a nitro-reducing pseudomonad ( Pseudomonas The strain MHY01 was deposited on May 23, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No: 34646.

[0014] This invention discovered that adding soil leachate can promote the absorption of thifluzamide by rice leaves and roots. Therefore, further screening of rhizosphere microbial strains in the soil leachate was conducted, and five rhizosphere microorganisms were isolated and identified. Among them, one rhizosphere microorganism, MHY01, had the effect of promoting the absorption of thifluzamide by rice and was identified as *Pseudomonas nitroreductoides*. Pseudomonas nitroreducens ), named Nitroreducing Pseudomonas strain MHY01 ( Pseudomonas nitroreducens strain MHY01 was deposited on May 23, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No: 34646, and classified as *Pseudomonas*.Pseudomonas (sp.), the address of which is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0015] The results of an indoor hydroponic experiment showed that, two days after inoculation with *Pseudomonas nitroreductoids*, the content of thifluzamide in rice roots (at an application concentration of 20 mg / L) increased by 26.99% compared to the control group, and three days later, it increased by 37.15% compared to the control group. This indicates that inoculation with *Pseudomonas nitroreductoids* can significantly increase the content of thifluzamide in rice roots.

[0016] This invention also provides a composite microbial community, wherein the composite microbial community consists of the above-mentioned nitro-reducing Pseudomonas MHY01 and Priestella megaterium (… Priestia megaterium ), Oligotrophosomes ( Stenotrophomonas pavanii Dendrocal microbes ( Microbacterium arborescens ) and Rhodococcus spp. ( Rhodococcus (sp.) constitutes the composition.

[0017] This invention also develops a compound microbial agent to promote pesticide absorption based on the regulatory effect of *Pseudomonas nitroreductoides* MHY01 on the absorption of thifluzamide in rice. The agent includes *Pseudomonas nitroreductoides* MHY01 and *Priscilla megaterium*. Priestia megaterium ), Oligotrophosomes ( Stenotrophomonas pavanii Dendrocal microbes ( Microbacterium arborescens ) and Rhodococcus spp. ( Rhodococcus Under hydroponic conditions, after inoculation with a compound microbial agent, the thifluzamide content in rice roots (at an application concentration of 20 mg / L) increased by 62.35% two days after inoculation with *Pseudomonas nitroreutonicus* alone, and by 48.17% three days later. Simultaneously, the compound microbial community had no effect on any of the rice's growth indicators. A pot experiment verified the effect of the compound microbial agent on thifluzamide absorption in rice, showing a 36.52% increase in root pesticide content on the third day. This indicates that the compound microbial community can enhance the absorption of systemic pesticides by plants, significantly increasing pesticide content in crops, thereby increasing pesticide utilization, improving pesticide control of crop diseases and pests, and reducing pesticide dosage.

[0018] Furthermore, the complex microbial community consists of equal volumes and concentrations of the aforementioned nitro-reducing Pseudomonas MHY01 and Priestella megaterium (…). Priestia megaterium ), Oligotrophosomes ( Stenotrophomonas pavanii Dendrocal microbes ( Microbacterium arborescens ) and Rhodococcus spp. ( Rhodococcus (sp.) constitutes the composition.

[0019] The present invention also provides a microbial agent comprising the above-mentioned nitro-reducing Pseudomonas MHY01 or the above-mentioned complex microbial community.

[0020] This invention overcomes the limitations of traditional pesticide use efficiency improvement technologies that rely on engineering methods by constructing artificial composite microbial communities. By revealing novel interaction mechanisms between functional strains and host crops, it innovatively establishes a new model for efficient pesticide utilization through "microbe-plant synergistic metabolism," enriching the resource pool of plant-microbe interactions promoting pesticide metabolism. This provides a new approach to overcoming the technical bottleneck of low plant absorption and transformation efficiency in existing pesticide residue management. The developed composite microbial agent not only significantly expands the functional microbial resource pool but also provides a novel technical path to solve the problem of pesticide residue blockage in the soil-crop system. Nitroreducing Pseudomonas MHY01 and its composite microbial agent demonstrate multiple application values ​​in improving pesticide utilization efficiency, reducing environmental residue risks, and constructing a farmland ecological restoration technology system, laying a crucial microbiome theoretical foundation and technical reserve for the development of green and safe agricultural chemical ecological regulation products.

[0021] Therefore, the present invention also provides the application of the above-mentioned nitro-reducing Pseudomonas MHY01, the above-mentioned complex bacterial group, or the above-mentioned bacterial agent as a thifluzamide synergist.

[0022] Furthermore, the thifluzamide synergist is used to promote the absorption of thifluzamide by plants. That is, the above-mentioned *Pseudomonas nitroreductoides* MHY01, the above-mentioned compound microbial community, or the above-mentioned microbial agent can be used as an absorption promoter to promote the absorption of thifluzamide by plants.

[0023] The present invention also provides a method for promoting the absorption of thifluzamide by plants, wherein the method comprises applying the above-mentioned Pseudomonas MHY01 or the above-mentioned compound bacterial group or the above-mentioned bacterial agent together with thifluzamide to the rhizosphere of rice.

[0024] By mixing Pseudomonas MHY01 or a compound bacterial agent with thifluzamide in rice culture medium, beneficial bacteria are delivered to the rhizosphere, thereby promoting the crop's absorption of pesticides, increasing the pesticide content in the crop, improving pesticide utilization, and enhancing control efficacy. Specifically, Pseudomonas MHY01 or a compound bacterial agent (final concentration OD) is added to the rice seedling culture medium. 600 =0.01) and thifluzamide (final concentration 20 mg / L), or by applying bacterial suspension (OD) through root irrigation. 600 =0.1). This method is easy to operate and suitable for large-scale planting scenarios. While ensuring the effectiveness of pest and disease control (such as improving the control effect of rice sheath blight), it reduces the amount of pesticides used, reduces the risk of soil and water pollution, alleviates the development of pesticide resistance, and promotes sustainable agricultural development.

[0025] Furthermore, the application method is to directly add hydroponic nutrient solution or to perform root irrigation treatment on soil-grown plants.

[0026] Furthermore, the final applied concentration of the strain, complex microbial community, or microbial agent is OD. 600 =0.01~0.1.

[0027] Furthermore, the plant in question is rice.

[0028] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a strain of *Pseudomonas nitroreductoids* MHY01 and its application as a thifluzamide synergist. This invention isolates a strain capable of promoting thifluzamide absorption in rice from healthy rice root soil, named *Pseudomonas nitroreductoids* MHY01, and further obtains a complex microbial community that promotes pesticide absorption. In practical applications, the strain and complex microbial community described in this invention, while ensuring pest and disease control efficacy, can significantly reduce pesticide soil adsorption loss, lower the risk of soil and water pollution, mitigate the development of pesticide resistance, and promote sustainable agricultural development. Secondly, these strains are all derived from the rhizosphere of healthy rice, are green and safe, and have no significant impact on rice growth (root length, plant height, and fresh weight showed no statistical difference compared to the control group), ensuring the quality and safety of agricultural products and ecological safety. Therefore, this invention, by developing rhizosphere microorganisms to improve pesticide utilization efficiency, can fundamentally solve the problem of "low utilization rate and increased dosage due to pesticide adsorption by soil." Attached Figure Description

[0029] Figure 1 The thifluzamide content in various parts of rice was compared with that in the control group using soil leachate. Figure 1 In the table, A represents the thifluzamide content in rice roots, and B represents the thifluzamide content in rice leaves.

[0030] Figure 2 These are microorganisms isolated from rice culture medium.

[0031] Figure 3 Phylogenetic tree of five rhizosphere soil microorganisms.

[0032] Figure 4 The growth curves are for five rhizosphere soil microorganisms.

[0033] Figure 5 The effects of five rhizosphere soil microorganisms on the uptake of thifluzamide by rice were investigated.

[0034] Figure 6 The effect of adding *Pseudomonas* on the thifluzamide content in rice roots was investigated. Figure 6 In the table, A represents the thifluzamide content in the rice roots on the second day; B represents the thifluzamide content in the rice roots on the third day.

[0035] Figure 7The effect of adding a compound microbial community on the thifluzamide content in rice roots was investigated. Figure 7 The left graph in the figure shows the thifluzamide content in rice roots on the first day; the middle graph shows the thifluzamide content in rice roots on the second day; and the right graph shows the thifluzamide content in rice roots on the third day. Note: Asterisks (*) in the figures indicate statistical significance levels: ns (P>0.05), * (P≤0.05), ** (P≤0.01), *** (P≤0.001), **** (P≤0.0001).

[0036] Figure 8 This study compares the effects of adding *Pseudomonas* and a complex bacterial flora on the thifluzamide content in rice roots. Among them, Figure 8 The left image shows the thifluzamide content in rice roots on the second day; the right image shows the thifluzamide content in rice roots on the third day.

[0037] Figure 9 The growth indicators of rice after inoculation with compound microbial communities are shown in the upper figure (the upper figure shows the aboveground part, and the lower figure shows the underground part).

[0038] Figure 10 The content of thifluzamide in rice roots was determined in a pot experiment with added compound microbial flora. Detailed Implementation

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0040] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0041] 1. Materials and Methods 1.1 Culture medium Trypticase (Tryptic) Soy Broth medium: contains 17 g tryptone, 3 g soybean peptone, 5 g sodium chloride, 2.5 g dipotassium hydrogen phosphate, 2.5 g glucose, and 1 L H2O. When preparing the solid medium, add 1.5% agar to the above medium.

[0042] Luria-Bertani medium: trypsin 10 g, yeast extract 5 g, NaCl 10 g, H2O 1 L. When preparing the solid medium, add 1.5% agar to the above medium.

[0043] All culture media were finally adjusted to pH 7 and then autoclaved at 121°C for 20 min.

[0044] 1.2 Rice culture medium: The conventional culture medium of the International Rice Research Institute (Yoshida) was used, and the formula is shown in Table 1.

[0045] Table 1. Formulas for Kimura B culture medium and International Rice Research Institute culture medium (mg / L)

[0046] 1.3 Preparation of Medicines and Reagents Thifluzamide technical grade: 98% purity, purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0047] Thifluzamide water-dispersible granules: active ingredient content 40%, registration certificate number: PD20150827, purchased from Shaanxi Huarong Kaiwei Biotechnology Co., Ltd.

[0048] Mass spectrometry grade methanol, mass spectrometry grade acetonitrile, mass spectrometry grade formic acid, purification agent N-propylethylenediamine (PSA), and graphitized carbon black (GCB) were all purchased from Shanghai Anpu Technology Co., Ltd.; sodium chloride was purchased from Tianjin Damao Chemical Reagent Factory.

[0049] Tryptone and yeast extract were purchased from OXOID and Beijing Qingke Biotechnology Co., Ltd.

[0050] Preparation of thifluzamide stock solution: Weigh 0.1 g of thifluzamide technical material, add acetonitrile (an organic solvent) to dissolve it completely, and finally dilute to 10 mL in a brown bottle. The concentration of the stock solution is 10000 mg / L.

[0051] 0.9% physiological saline: Accurately weigh 9 g NaCl, add 1000 mL of distilled water, stir with a glass rod to make a complete solution, pour into a volumetric flask, seal and sterilize at high temperature.

[0052] 0.2% formic acid aqueous solution: Add 2 mL of formic acid and dissolve it in 1000 mL of triple-distilled water.

[0053] 1.4 Test instruments and equipment (Table 2) Table 2. Main test instruments and their manufacturers

[0054] 1.5 Test Crops and Soil The test crop was rice, variety Nipponbare, purchased from Xiantao Ruideweien Technology Co., Ltd.

[0055] The soil samples used in the pot experiment were all collected from the Zengcheng Experimental Base of South China Agricultural University. During the collection process, soil samples were randomly collected from 5 to 20 cm depths, and then dried, crushed, and sieved (8 mesh) to remove plant impurities, animal remains, and other impurities.

[0056] 1.6 List of tested strains (see Table 3) Table 3 List of strains

[0057] Example 1: Effects of rhizosphere microbiota on the uptake and utilization of thifluzamide in rice I. Experimental Methods 1. To screen for specific functional bacteria that can effectively promote the absorption of thifluzamide in rice, this invention employs the following core steps to initiate the screening process: Constructing a simulated enrichment system: Fresh soil samples (0-15 cm topsoil) were collected from the farmland system of the Zengcheng Experimental Base of South China Agricultural University in Guangdong Province. 5 g of soil was weighed and added to a 250 mL Erlenmeyer flask containing 100 mL of sterile distilled water, and the mixture was thoroughly shaken. The resulting soil leachate was filtered through filter paper to obtain a soil extract. In a controlled hydroponic rice cultivation system, 10 mL of the soil extract was added to Yoshida rice culture medium containing 20 mg / L thifluzamide and co-cultured with rice to introduce a complex soil microbial community as a potential source of absorption-promoting bacteria. A control group was prepared by sterilizing the soil leachate through a 0.22 μm filter membrane. Sampling at target time points: Sampling was conducted on days 0, 1, 3, 5, and 7 to extract thifluzamide from rice roots and leaves, and the results were detected by LC-MS.

[0058] 2. Thifluzamide detection method (1) Detection was performed using a Waters ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS) system with a Waters ACQUITY UPLC column. Ⓡ A BEH C18 column (2.1 mm × 50 mm, 1.7 μm) was used at a column temperature of 40 °C, a flow rate of 0.3 mL / min, and an injection volume of 5 μL. The mobile phase consisted of 0.2% formic acid (A) and methanol (D), and gradient elution was used (program shown in Table 4). Mass spectrometry was performed using an XEVO TQD triple quadrupole in negative ion mode with an electrospray ionization (ESI) source. Multiple reaction monitoring (MRM) was employed with a capillary voltage of 3.0 kV, a cone voltage of 30 V, a source temperature of 120 °C, a desolvation gas temperature of 350 °C, and a desolvation gas flow rate of 550 L / Hr. The thifluzamide mass spectrometry detected a precursor ion of 526.8 m / z and daughter ions of 165.9 and 124.9 m / z, with collision energies of 24 and 45 V, respectively. The 526.8 / 165.9 m / z ion pair was used for quantitative analysis.

[0059] Table 4. Mobile phase ratio and flow rate for UPLC detection

[0060] (2) Standard curve and linear range The concentration of thifluzamide was determined using the external standard method, and the concentration was correlated with the peak area in the liquid phase using a univariate linear equation. The thifluzamide stock solution was diluted with acetonitrile to prepare six concentrations: 100, 50, 10, 1, 0.5, and 0.1 mg / L. A standard curve was plotted with peak area as the ordinate and standard solution concentration as the abscissa, and a regression equation was fitted.

[0061] 3. Extraction methods of thifluzamide from different samples (1) Soil: Accurately weigh 5 g of soil into a 50 mL centrifuge tube, add 10 mL of mass spectrometer acetonitrile to the centrifuge tube and vortex for 1 min, then place it in an ultrasonic cleaner and sonicate for 30 min, add 3 g of sodium chloride, vortex for 1 min, centrifuge at 5000 rpm at room temperature for 3 min, use a disposable syringe to draw the supernatant from the centrifugation, filter it through a 0.22 µm microporous organic filter membrane and collect it in a brown sample bottle for testing.

[0062] (2) Rice culture medium: Accurately measure 3 mL of culture medium into a 10 mL centrifuge tube using a pipette, add 3 mL of mass spectrometer acetonitrile to the centrifuge tube and vortex for 1 min, then place it in an ultrasonic cleaner and sonicate for 30 min, add 3 g of sodium chloride, vortex for 1 min, centrifuge at 5000 rpm at room temperature for 3 min, use a disposable syringe to draw the supernatant from the centrifugation, filter it through a 0.22 µm microporous organic filter membrane and collect it in a brown sample bottle for analysis.

[0063] (3) Rice leaves: Accurately weigh 0.5 g of sample into a 5 mL grinding tube, add grinding beads, add 3 mL of mass spectrometer acetonitrile, grind thoroughly, and then extract by ultrasonication for 30 min. Add 1.0 g of sodium chloride, shake for 1 min, and centrifuge at 9000 rpm for 3 min. Take 1 mL of supernatant into a 2 mL test tube containing 50 mg PSA and 10 mg GCB, shake for 1 min, centrifuge at 10000 rpm for 5 min, and use a disposable syringe to draw the supernatant, filter it through a 0.22 µm microporous organic filter membrane, and collect it in a brown sample bottle for analysis.

[0064] (4) Rice roots: Accurately weigh 0.2 g of sample into a 5 mL grinding tube, add grinding beads, add 3 mL of mass spectrometer acetonitrile, grind thoroughly, and then extract by ultrasonication for 30 min. Add 1.0 g of sodium chloride, shake for 1 min, and centrifuge at 9000 rpm for 3 min. Take 1 mL of supernatant into a 2 mL test tube containing 50 mg of PSA, shake for 1 min, centrifuge at 10000 rpm for 5 min, and use a disposable syringe to draw up the supernatant, filter it through a 0.22 µm microporous organic filter membrane, and collect it in a brown sample bottle for analysis.

[0065] 4. Determination of the recovery rate of thifluzamide addition Take soil, culture medium, rice leaves and roots that are not contaminated by pesticides, weigh them into 50 mL centrifuge tubes according to the quality requirements of each component, and add 10 μL of thifluzamide standard solution of different concentrations to the centrifuge tubes so that the final concentration of thifluzamide in the soil reaches 50, 5 and 0.5 mg / L, the final concentration of thifluzamide in the culture medium reaches 50, 5 and 0.5 mg / L, the final concentration of thifluzamide in the rice leaves reaches 100, 10 and 1 mg / kg, and the final concentration of thifluzamide in the rice roots reaches 1000, 100 and 10 mg / kg, respectively. Extract and determine the concentrations according to the above method, and calculate the recovery rate.

[0066] II. Experimental Results Experimental results are as follows Figure 1 As shown, after the addition of soil leachate, the roots showed significant differences compared to the control group on the third day, and the leaves showed significant differences on the third and fifth days. Therefore, it is speculated that there are bacteria in the soil leachate that can promote the absorption of thifluzamide by rice.

[0067] Example 2: Isolation, screening, and characterization of rhizosphere microbial strains I. Experimental Methods 1. Enrichment and domestication of rhizosphere microbial strains Fresh soil samples were collected from the farmland system (0-15 cm topsoil) of the Zengcheng Experimental Base of South China Agricultural University in Guangdong Province. 5 g of soil was weighed and added to a 250 mL Erlenmeyer flask containing 100 mL of sterile distilled water. The mixture was thoroughly shaken and the resulting soil leachate was filtered through filter paper to obtain a soil extract. 10 mL of this soil extract was added to a Yoshida rice culture medium containing 20 mg / L thifluzamide. The rice was co-cultured for 14 days. 10 mL of this culture medium was then added to another Yoshida rice culture medium containing 20 mg / L thifluzamide. After multiple passages, the thifluzamide content in rice cultured with and without the soil extract was measured to obtain a microbial community that may have a potential absorption-promoting function.

[0068] 2. Purification and isolation of rhizosphere microorganisms Take 1 mL of rice culture broth after multiple passages and transfer it to 9 mL of physiological saline, then dilute it serially to a 10⁻⁶ level. -2 10 -3 10 -4 10 -5 10 -6 10 -7 Then, take 10. -4 ~10 -7100 μL of the liquid was inoculated onto TSA solid medium. After 48 h of incubation, single colonies were picked from the plates and streaked to isolate the purified strain. The purified strain was then inoculated onto TSB medium for scale-up culture to determine its ability to promote thifluzamide uptake in rice. Finally, the strain exhibiting the ability to promote thifluzamide uptake in rice was stored in glycerol tubes at -80°C.

[0069] 3. Molecular identification of 16S rRNA in rhizosphere microorganisms The refrigerated degradation strains obtained from Method 2 above, which enhance the absorption-promoting effect, were reactivated and revived by taking 1 mL of fresh bacterial culture. Total microbial DNA was extracted according to the instructions of the microbial genomic DNA extraction kit.

[0070] Using the extracted total DNA as a template, universal bacterial primers were used, with the upstream primer being 27F (5'-TACGGTTACCTTGTTACGACTT-3') and the downstream primer being 1492R (5'-AGAGTTTGATCCTGGCTCAG-3').

[0071] The PCR reaction volume was 25 μL, including 2 μL of 27F upstream primer, 2 μL of 1492R downstream primer, 1 μL of DNA template, 12.5 μL of mixed enzyme (Taq enzyme, dNTPs, MgSO4, etc.), and 7.5 μL of ddH2O. PCR reaction conditions: denaturation at 94℃ for 3 min; 35 cycles of 94℃ for 30 s, 54℃ for 30 s, 72℃ for 1 min, followed by extension at 72℃ for 10 min.

[0072] After PCR amplification, the product was purified, and 16S sequencing was performed by Sangon Biotech. The obtained 16S rRNA sequence was compared with the microbial 16S database sequence from NCBI (https: / / www.ncbi.nlm.nih.gov / ) using BLAST. After homology comparison analysis, the relevant microbial 16S rRNA sequences with high homology were downloaded. Alignment analysis was performed using Clustal X 1.8.1, and after neatly cutting the sequences at both ends, a phylogenetic tree was constructed using the Neighbor-Joining algorithm in MEGA 7.0.

[0073] 4. Observation of rhizosphere microbial morphology Draw 0.2 mL of liquid from the glycerol tube and transfer it to fresh LB liquid medium for activation. When the growth reaches the logarithmic phase, use an inoculation stick to pick up the bacterial solution and streak it onto TSA solid medium. Incubate at 30°C. After single colonies appear on the plate, observe the morphological characteristics of the colonies, such as color, shape, size, and transparency.

[0074] 5. Determination of the growth curve of the complex microbial community For the five rhizosphere microorganisms collected, single colonies after isolation and purification were picked and cultured in TSB medium for 12 h. The resulting bacterial suspension was centrifuged at 4000 r / min, the supernatant was discarded, and the suspension was washed with PBS and resuspended to achieve OD. 600 A value of 1 was used as the seed culture. The inoculum was transferred to freshly sterilized TSB medium, with three replicates. The culture was incubated in a constant temperature shaker at 30°C and 180 rpm. The OD value of the culture was measured every 2 h. 600 Measurements were performed up to 60 hours later. The result was measured using OD... 600 The values ​​are plotted on the ordinate and the culture time on the abscissa to create a growth curve for the strain.

[0075] II. Experimental Results 1. After enrichment, domestication, isolation, and purification, a total of 10 purified strains capable of colonizing rice Yoshida broth were obtained from the rhizosphere microbial strains. The results are as follows: Figure 2 As shown.

[0076] 2. Identification of 16S rRNA in rhizosphere microbial strains DNA was extracted from the enriched total microorganisms. Using the extracted total microbial DNA as a template, PCR amplification was performed using universal bacterial primers (27F, 1492R). The amplified products were then sequenced, and the sequencing results were trimmed. The obtained 16S rRNA fragments were 1428 bp, 1444 bp, 1441 bp, 1446 bp, and 1444 bp in size. Blast analysis was performed on the sequences in the NCBI database. Highly homologous sequences were downloaded and analyzed using Clustal X 1.8.1 and the Neighbor-Joining algorithm in MEGA 7.0 to construct a phylogenetic tree. Figure 3 As shown, the 16S rRNA sequences of rhizosphere microbial strains are respectively compared with those of Pseudomonas spp. in NCBI (…). Pseudomonasnitroreducens strain DSM14399), Priestella spp. Priestia sp.), Oligotrophomonas genus ( Stenotrophomonas sp.), Microbacterium spp. Microbacteriumarborescens strain DSM 20754), Rhodococcus ( Rhodococcus sp. The bacteria exhibit high similarity, with homology exceeding 99%. Combined with morphological observation, the strains can be preliminarily identified as *Pseudomonas nitroreductoids* (…). Pseudomonasnitroreducens ), Priestella spp. Priestia sp.), Oligotrophomonas genus ( Stenotrophomonas sp.), dendritic microbes ( Microbacteriumarborescens), Rhodococcus ( Rhodococcus sp.).

[0077] 3. Growth and development curves of rhizosphere microbial strains By analyzing 60 h microbial OD 600 The values ​​were detected, and the growth curves of five rhizosphere microbial strains were obtained, such as... Figure 4 As shown. The rhizosphere thifluzamide-absorbing bacteria exhibit a lag phase of 0–6 h, during which they accumulate sufficient enzymes and energy for cell volume increase, metabolic activity, and cell proliferation. The logarithmic growth phase occurs from 6–20 h, during which the bacteria rapidly consume nutrients in the culture medium for rapid metabolic activity and proliferation. The stationary phase occurs from 20–60 h, during which the bacterial reproduction rate decreases due to nutrient depletion and the accumulation of toxic substances produced by metabolism. The OD values ​​for the four bacteria at the stationary phase are as follows: 600 The value is approximately 1.2.

[0078] 4. The effect of bacterial flora on the absorption of thifluzamide in rice was tested, and the results are as follows: Figure 5 As shown, *Pseudomonas nitroreductoides* MHY01 has the ability to promote the absorption of thifluzamide in rice. Gram staining of MHY01 confirmed it to be a Gram-negative bacterium. On TSA medium, its colonies appear as pale white, raised lesions with irregular edges, and bacterial growth along the edges. The colonies are composed of single, straight or curved bacilli with polar flagella (see Table 5), further confirming that this strain is *Pseudomonas nitroreductoides*. Pseudomonas nitroreducens strain MHY01). This strain was deposited on May 23, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No: 34646, and classified as *Pseudomonas*. Pseudomonas sp., the address of which is No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0079] Table 5 Results of cell morphology assay

[0080] 5. Physiological and biochemical characteristics of MHY01 Different microorganisms possess different enzyme systems and varying abilities to decompose nutrient substrates, resulting in different metabolic products that can be used to identify bacteria. Biochemical tests are used to detect the metabolic effects of bacteria on various substrates and their metabolic products, thereby identifying bacterial species. The main physiological and biochemical characteristics of the pesticide absorption-promoting bacterium MHY01 are as follows (Table 6).

[0081] Table 6 Results of the test on the utilization of the sole carbon source

[0082] In summary, this invention isolated and identified five specific rhizosphere microorganisms from soil leachate. One of these microorganisms, MHY01, exhibited the effect of promoting the absorption of thifluzamide in rice and was identified as *Pseudomonas nitroreductoides*. Pseudomonas nitroreducens ), named Nitroreducing Pseudomonas strain MHY01 ( Pseudomonas nitroreducens To further investigate whether four other strains that could not promote the absorption of thifluzamide in rice could further enhance the promoting effect of MHY01, this invention utilizes four specific strains. Priestia megaterium ATCC 14581 、Stenotrophomonas pavanii (GDMCC NO.: 802177) 、 Microbacterium arborescens CICC 20195 、Rhodococcus After mixing sp. CICC 10278 and MHY01 in equal proportions (equal volume and concentration), the mixture was cultured in TSB medium for 24 h, washed with PBS and resuspended to allow OD to reach the target concentration. 600 A value of 1 is used to prepare a compound bacterial agent.

[0083] Example 3: Effects of rhizosphere absorption-promoting bacteria on the absorption of thifluzamide in rice I. Experimental Methods 1. Pot experiment Rice seeds were sown in seedling trays and the seedlings were raised to the three-leaf stage (approximately 10 cm in height). 850 g of sieved, dry soil was weighed and mixed with 1 mL of 18000 mg / L thifluzamide and 49 mL of distilled water in an Erlenmeyer flask. This mixture was then added to the soil and stirred until the initial thifluzamide concentration in the soil reached 20 mg / kg. The mixed soil was then placed in flowerpots (13.5 cm in diameter and 13 cm in depth). Six uniformly growing rice seedlings were selected and transplanted into the flowerpots, with six seedlings planted per treatment. All flowerpots were placed in an artificial climate chamber for cultivation, and soil moisture was checked regularly and watered artificially to maintain humidity. The experiment included a control (CK) group, a Pseudomonas aeruginosa treatment group, and a compound microbial community treatment group (the compound microbial agent was the same as in Example 2): after rice transplanting, the bacterial suspension (50 mL, OD500 mg / kg) was applied to the roots via root irrigation. 600 A 0.1% concentration was applied to the roots of rice plants, with six replicates for each treatment. Samples were collected 1, 2, and 3 days after rice transplanting, including samples from rice plants and soil from the root zone. The rice roots were washed four times with distilled water to ensure complete removal of any adhering soil and pesticide. The plant surfaces were dried with filter paper, and then the pesticide thifluzamide was extracted from the rice plants and soil, and its concentration was determined.

[0084] 2. Hydroponic Experiment Rice seeds were sown in seedling trays and raised to the three-leaf stage (approximately 10 cm in height). 850 mL of Yoshida rice culture solution was measured, and 1 mL of 18000 mg / L thifluzamide preparation and 49 mL of distilled water were added to an Erlenmeyer flask. After mixing, this mixture was added to the Yoshida rice culture solution and mixed thoroughly to achieve an initial thifluzamide concentration of 20 mg / kg. The prepared culture solution was transferred to hydroponic boxes, and six uniformly growing rice seedlings were transplanted into each box. Six seedlings were planted per treatment. All hydroponic boxes were placed in an artificial climate chamber and watered regularly. The experiment included a control (CK) group, a Pseudomonas treatment group, and a compound microbial treatment group (the compound microbial agent was the same as in Example 2). After rice transplanting, the microbial suspension was applied to the rice roots via root irrigation, ensuring the final OD of the microbial agent in the rice culture solution reached a certain level. 600 The threshold value was 0.01, and each treatment was performed in 6 replicates. Samples were collected 1, 2, and 3 days after rice transplanting, including rice plant samples and rice culture medium. The rice roots were washed four times with distilled water to ensure complete removal of any pesticide adhering to the root surface. The plant surface was dried with filter paper, and then the pesticide thifluzamide in the rice plants and culture medium was extracted and its content was determined.

[0085] 3. Measurement of root elongation, plant height, and fresh weight. Ten whole rice plants were randomly collected, washed with deionized water, and blotted dry with filter paper. The rice plants were then separated into aboveground and root parts. The root elongation and plant height of each treatment were measured using a ruler (root elongation refers to the distance between the tip of the longest root and the rhizome, and plant height refers to the distance between the rhizome and the top of the main stem). The fresh weight of the aboveground and root parts of the rice was measured using an analytical balance.

[0086] II. Experimental Results 1. Effects of microbial inoculants on the absorption of thifluzamide by rice roots under hydroponic conditions To investigate the impact of rhizosphere microbiota on pesticide absorption, bacterial solutions of different rhizosphere strains were added to the roots of rice plants. The differences in thifluzamide concentrations in rice plants treated with different rhizosphere bacteria were compared to clarify the influence of rhizosphere-promoting bacteria on pesticide absorption in rice. Figure 6 It can be seen that, compared with the blank control group, the thifluzamide content in rice roots increased by 26.99% and 37.15% respectively after 2 and 3 days of Pseudomonas inoculum treatment; Figure 7 It can be seen that after treatment with the compound microbial community, the thifluzamide content in rice roots increased by 74.35%, 106.17%, and 103.21% at 1 day, 2 days, and 3 days, respectively; Figure 8It was found that 2 days and 3 days after the bacterial suspension treatment, the effect of the compound microbial community on promoting the absorption of thifluzamide in rice was increased by 62.35% and 48.17% compared with the use of Pseudomonas alone. This indicates that Pseudomonas can promote the absorption and utilization of thifluzamide in rice, and the compound microbial community can significantly improve the promoting effect of Pseudomonas.

[0087] 2. The effects of microbial inoculants on rice growth under hydroponic conditions To clarify the potential effects of this complex microbial community on the physiological traits of rice, key growth indicators such as root length, plant height, aboveground fresh weight, and underground fresh weight of rice seedlings were systematically measured and compared in simultaneous experiments with control and treatment groups. Figure 9 As shown, there were no statistically significant differences in rice growth indicators between the treatment group and the control group during the observation period.

[0088] This compound microbial community did not exhibit growth-promoting effects, which is significantly different from the functional characteristics of traditional plant rhizosphere growth-promoting bacteria (PGPR), proving that its physiological impact on rice growth is neutral. This result further indicates that the effect of the compound microbial community in enhancing pesticide absorption efficiency is independent of crop growth regulation pathways, and its mechanism of action is fundamentally different from existing microbial fertilizers or biostimulants. While ensuring pesticide efficacy enhancement, the microbial community avoids interfering with normal crop growth and development, providing a new pathway for developing specific pesticide-enhancing microbial preparations.

[0089] 3. Effects of microorganisms on the uptake of thifluzamide by rice roots under pot cultivation conditions The content of thifluzamide in rice roots was compared at 1, 2, and 3 days after treatment under pot conditions to clarify the effect of microbial inoculants on pesticide absorption in rice. After thifluzamide application, the concentration of thifluzamide in rice roots was significantly higher than that in the control group starting from day 2. Figure 10 Compared to the blank control group, the compound microbial inoculant treatment increased the thifluzamide content in rice roots by 50.28% on the second day after application and by 36.52% on the third day. The addition of microbial inoculants significantly increased the thifluzamide content in rice roots, indicating that the compound microbial inoculant can promote the absorption and utilization of thifluzamide by rice.

[0090] In summary, the nitro-reducing Pseudomonas MHY01 strain of the present invention ( Pseudomonas nitroreducens Both strain MHY01 and the complex microbial community can enhance the absorption of systemic pesticides by plants, thus significantly increasing the pesticide content in crops, thereby increasing pesticide utilization, improving the control effect of pesticides on crop diseases and pests, and reducing the amount of pesticides used.

Claims

1. A strain of nitro-reducing Pseudomonas ( Pseudomonas sp.)MHY01, characterized in that, The nitro-reducing Pseudomonas was deposited on May 23, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No: 34646.

2. A complex microbial community, characterized in that, The complex microbial community consists of *Pseudomonas megaterium* MHY01 and *Priscilla megaterium* as described in claim 1. Priestia megaterium ), Oligotrophosomes ( Stenotrophomonas pavanii Dendrocal microbes ( Microbacterium arborescens ) and Rhodococcus spp. ( Rhodococcus (sp.) constitutes the composition.

3. The complex microbial community according to claim 2, characterized in that, The complex microbial community consists of equal volumes and concentrations of *Pseudomonas megaterium* MHY01 and *Priscilla megaterium* as described in claim 1. Priestia megaterium ), Oligotrophosomes ( Stenotrophomonas pavanii Dendrocal microbes ( Microbacterium arborescens ) and Rhodococcus spp. ( Rhodococcus (sp.) constitutes the composition.

4. A microbial agent, characterized in that the microbial agent comprises the nitro-reducing Pseudomonas MHY01 of claim 1 or the complex microbial community of any one of claims 2 or 3.

5. The use of the Nitroreducing Pseudomonas MHY01 of claim 1, the complex microbial community of claim 2 or 3, or the microbial agent of claim 4 as a thifluzamide synergist.

6. The application according to claim 5, characterized in that, The thifluzamide synergist is used to promote the absorption of thifluzamide by plants.

7. A method for promoting the absorption of thifluzamide by plants, characterized in that, The method involves applying the nitro-reducing Pseudomonas MHY01 of claim 1, any of the complex microbial groups of claim 2 or 3, or the microbial agent of claim 4, together with thifluzamide to the plant rhizosphere.

8. The method according to claim 7, characterized in that, The application method is to directly add hydroponic nutrient solution or to perform root irrigation treatment on soil-grown plants.

9. The method according to claim 7, characterized in that, The final applied concentration of the strain, complex flora, or inoculant is OD. 600 =0.01~0.

1.

10. The application according to claim 6 or the method according to claim 7, characterized in that, The plant in question is rice.

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