Application of Streptomyces fradiae fermentation broth in promotion of degradation of amide herbicides and / or improvement of soil microbial community structure

Abstract

The invention discloses application of streptomyces fradiae fermentation liquor in any one or more of the following items. (1) degradation of amide herbicides in soil is promoted; and (2) improving the community structure of soil microorganisms, namely reducing the abundance of L.virens and increasing the abundance of Buddmonas, proteobacteria and functional bacteria. The streptomyces fradiae fermentation liquor effectively relieves the influence of tobacco phytotoxicity caused by compound residues of the amide herbicides through dual mechanisms of synergistic degradation and improvement of abundance of microbial communities, and a theoretical basis and a technical support are provided for bioremediation of the amide herbicides in farmland.

Description

Technical Field

[0001] This invention relates to the application of a Streptomyces freundii fermentation broth in promoting the degradation of amide herbicides and / or improving the structure of soil microbial communities, belonging to the field of amide herbicide degradation technology. Background Technology

[0002] Tobacco is an important economic crop. With the advancement of agricultural production technology, chemical weeding has become a widely used and indispensable field management measure for tobacco, leading to a continuous increase in herbicide use and farmers' growing dependence on it. However, improper use of herbicides can easily cause phytotoxicity risks, threatening tobacco yield and quality safety. Tobacco herbicide damage is mainly divided into two categories: one is direct phytotoxicity caused by improper application during the current season, and the other is secondary residual phytotoxicity caused by the application of long-acting residual herbicides in the previous crop.

[0003] Currently, common amide herbicides include acetochlor, metolachlor, butachlor, propargite, alachlor, and bensulfuron-methyl. Acetochlor, butachlor, and metolachlor account for 96% of the global market share of amide herbicides, making them the three leading products in this category. These herbicides effectively suppress the growth of annual grasses and common broadleaf weeds, and have been widely used in the production and planting of major crops such as corn and cotton, becoming a key plant protection method in agricultural production. Acetochlor was listed as a Group 2B carcinogen by the U.S. Environmental Protection Agency in 2008.

[0004] In tobacco cultivation, improper use of amide herbicides can cause various types of phytotoxicity: mild cases result in rough, wrinkled leaf surfaces, hindered leaf unfolding, and curling and chlorosis from leaf tip to leaf margin, leading to stunted growth and shortened internodes; severe cases cause irreversible damage such as deformed and scorched leaves, browning and death of stems and leaves, seriously affecting the normal growth and development of tobacco plants. Mild phytotoxicity allows plants to recover slowly through their own growth, but severe phytotoxicity leads to yield reduction. The applicant's preliminary field trials showed that residues of three amide herbicides (acetochlor, metolachlor, and bensulfuron-methyl) significantly inhibited tobacco growth, inducing typical phytotoxicity symptoms such as leaf yellowing and malformation as early as 14 days after transplanting. Furthermore, with prolonged application time (42 days after transplanting), a very prominent dose-dependent inhibitory effect was observed. Acetochlor showed the most significant phytotoxicity effect on tobacco, specifically manifested as yellow-brown lesions on leaves, chlorotic halos on leaf margins, and rat-tail-like deformities. When the application rate reached the recommended dose, the plants exhibited severe growth impairment. Based on the dose-inhibition rate, the critical concentration for phytotoxicity was calculated to be 84.38 g ai / ha (1 / 16R). The phytotoxicity symptoms of metolachlor were similar to those of acetochlor, but to a lesser degree. Although its plant height inhibition rate was higher than its leaf length inhibition rate, its overall inhibitory effect was weaker than that of acetochlor. Experiments calculated the critical phytotoxicity concentration for metolachlor to be 101.25 g ai / ha (1 / 16R). The phytotoxicity characteristics of bensulfuron-methyl were mainly manifested by leaf yellowing and whitening, with a critical phytotoxicity concentration of 93.75 g ai / ha (1 / 8R).

[0005] Currently, the main degradation mechanisms of amide herbicides fall into three categories: chemical, physical, and microbial degradation. Chemical degradation primarily includes oxidation, reduction, hydrolysis, and the formation of insoluble salts or complexes with other substances. Studies have found that FeS2 treated with steam heat increased the degradation rate of metolachlor by 23 times compared to untreated material; the addition of alginate significantly accelerated the degradation rate of metolachlor, reducing its concentration to below 50% of its initial value after 5 days; and after 1 hour of irradiation with a 150W xenon lamp, the TOC removal rate of metolachlor using a graphene / TNAs photoelectrode reached 64.63%. Physical degradation methods mainly include adsorption removal and ultrasonic washing. In soil environments, adsorption is a key process determining herbicide migration, transformation, and bioavailability. Studies show that Cu... 2+ Cr 6+ and Zn 2+Kaolinite can enhance the adsorption capacity of acetochlor. When the concentration of acetochlor is below 2 mg / L, the effect of metal ions on its adsorption is not significant; however, when the concentration exceeds 5 mg / L, the promoting effect of metal ions becomes more significant. Furthermore, sodium-type montmorillonite and organically modified montmorillonite exhibit good adsorption performance for acetochlor, while organic clay is more effective in removing acetochlor from soil and water. Regarding ultrasonic technology, studies have shown that when the ultrasonic power is 1900 W, the acetochlor concentration decreases rapidly within the first 20 minutes and tends to stabilize after 60 minutes, achieving a removal rate of 83.22%. Microbial degradation is the main pathway for herbicide decomposition; fungi, bacteria, and actinomycetes decompose pesticides through secretion of metabolites or direct action. Hou Jiawen screened two metolachlor-degrading bacteria from farmland soil: *Trichoderma harzianum* achieved a 96.1% degradation rate of 50 mg / L metolachlor with a half-life of 4.07 days at pH 6.73, an inoculum size of 564 mg / L, and 26.8℃; *Phyllobacterium tumefaciens* achieved a 76.4% degradation rate with a half-life of 5.88 days at pH 5.55, an inoculum size of 9.7%, and 29.2℃. Wang et al. found that *Bacillus cereus* RM2, *Bacillus thuringiensis* MA3, and *Bacillus* sp. Met1 achieved a degradation rate of over 80% for metolachlor and approximately 50% for both propachlor and pretilachlor. Ni Yingying's team isolated *Paragonimus* strain Y3B-1 from an acetochlor wastewater treatment system. This strain achieved an 86.7% degradation rate of acetochlor at 30℃ and pH=7, with the degradation efficiency positively correlated with the inoculum size. Tian Shuang et al. obtained *Mallotus* strain WN-3, which achieved a 38.3% degradation rate of acetochlor at 35℃ and pH=6. Dong Bin et al. isolated *Streptococcus adhesioides* A-3, which achieved a 33.6% degradation rate of 10 mg / L acetochlor; the addition of glucose and NaCl improved the degradation efficiency. Luo et al. identified *Pseudomonas aeruginosa* JD115, which rapidly degraded acetochlor at 37℃ and pH=7, achieving a degradation rate of 95.4% after nutrient supplementation. Ni Jun et al. reported that *Synthia schenckii* Y-4 achieved an 83.3% degradation rate of 50 mg / L acetochlor within 48 hours at 30℃ and pH=8. Studies have shown that *Penicillium marquandii* can degrade 500 mg / L metolachlor by 96.3% within 7 days. Saxena et al.'s research indicated that *Actinomycetes* sp. can degrade 100 mg / L metolachlor by 60% within 27 days.

[0006] However, the aforementioned strains were mainly isolated from contaminated soil, water bodies, and crops, but most of the related findings are limited to the laboratory research stage and have not yet been industrialized for large-scale field application. Currently, the functions of commercially available microbial agents are mostly focused on improving fertilizer efficiency and promoting root and seedling growth, while research on their degradation effects on amide herbicides is relatively limited. Summary of the Invention

[0007] Based on the above, the present invention provides an application of *Streptomyces freundii* fermentation broth in promoting the degradation of amide herbicides and / or improving the structure of soil microbial communities.

[0008] The technical solution of the present invention is: the application of *Streptomyces freundii* fermentation broth in any one or more of the following:

[0009] (1) Promotes the degradation of amide herbicides in the soil;

[0010] (2) Improve the community structure of soil microorganisms, including reducing the abundance of Chlorophyta and increasing the abundance of Bacillus, Proteobacteria and functional genera.

[0011] Preferably, the *Streptomyces freundii* fermentation broth is prepared by mixing *Streptomyces freundii* agent, a fermentation-type carbon source, and water, fermenting in the dark, and then diluting before use.

[0012] Preferably, the Streptomyces freundii agent, fermentable carbon source, and water are mixed in a mass ratio of 1:5:100.

[0013] Preferably, the amide herbicide is any one or more of acetochlor, metolachlor, and bensulfuron-methyl.

[0014] The beneficial effects of this invention are as follows: *Streptomyces freundii* fermentation broth significantly enhances the degradation of amide herbicides, significantly increases soil microbial diversity index, regulates microbial community structure and function, and strengthens soil ecological stability and stress resistance. In particular, *Streptomyces freundii* fermentation broth effectively mitigates the effects of tobacco phytotoxicity caused by the combined residues of amide herbicides through a dual mechanism of synergistic degradation and increased microbial community abundance, providing a theoretical basis and technical support for the bioremediation of amide herbicides in farmland. Attached Figure Description

[0015] Figure 1 Agronomic traits of tobacco at 30 days after transplanting in herbicide control and blank control treatments (**** indicates significance level less than 0.0001).

[0016] Figure 2 Comparison of tobacco agronomic traits at 30 days after transplanting between treatments with fermentation broth of six microbial agents and treatments with direct application of diluted microbial agents (ns indicates no significant difference; * indicates a significance level less than 0.05).

[0017] Figure 3 Agronomic traits of tobacco treated with fermentation broths of six microbial agents 30 days after transplanting

[0018] Figure 4 Agronomic traits of tobacco at 70 days after transplanting in herbicide control and blank control treatments (**** indicates significance level less than 0.0001).

[0019] Figure 5 Maximum leaf length at 70 days after transplanting for treatment with fermentation broths of six microbial agents (*** indicates significance level less than 0.001; **** indicates significance level less than 0.0001)

[0020] Figure 6 Maximum leaf width at 70 days after transplanting for treatment with fermentation broths of six microbial agents (ns indicates no significant difference; * indicates significance level less than 0.05; *** indicates significance level less than 0.001; **** indicates significance level less than 0.0001)

[0021] Figure 7 Plant height at 70 days after transplanting treated with fermentation broths of six microbial agents (**** indicates significance level less than 0.0001).

[0022] Figure 8 Stem circumference at 70 days after transplanting treated with fermentation broths of six microbial agents (** indicates significance level less than 0.01; **** indicates significance level less than 0.0001)

[0023] Figure 9 Overlap of bacterial communities in soil after treatment with fermentation broths of different microbial agents (BM, BS, CKY, PL, Pf, SF, and TL represent Bacillus megaterium, Bacillus subtilis, herbicide control, Paecilomyces lilacinus, Pseudomonas fluorescens, Streptomyces freundii, and Trichoderma longifolia, respectively).

[0024] Figure 10 Top 10 dominant bacterial community structure species abundance at the phylum level

[0025] Figure 11 Species abundance of the top 10 dominant bacterial communities at the genus level relative abundance

[0026] Figure 12 Heatmap of relative abundance of the top 20 bacteria at each treatment level

[0027] Figure 13 Degradation trends of acetochlor in each treatment group

[0028] Figure 14 Degradation trends of metolachlor in each treatment group

[0029] Figure 15 Degradation trends of bensulfuron-methyl in each treatment group. Detailed Implementation

[0030] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0031] I. Test Time and Location

[0032] 1. Trial period: April-October 2024

[0033] 2. Test location: Kedu Town, Pingtang County, Qiannan Buyi and Miao Autonomous Prefecture, Guizhou Province

[0034] II. Test reagents and crops

[0035] 1. Test pesticide:

[0036] 72% Metolachlor EC (PD2008356): Shandong Qiaochang Modern Agriculture Co., Ltd.;

[0037] 900g / L acetochlor emulsifiable concentrate (PD20100119): Shandong Aokun Crop Science Co., Ltd.;

[0038] 50% Benthiamethoxam Wettable Powder (PD20070377): Jiangsu Kuida Agrochemical Co., Ltd.

[0039] 2. Test inoculum:

[0040] Trichoderma longifolia powder (live bacteria count ≥ 200 million / g): Henan Haomeite Biotechnology Co., Ltd.;

[0041] Fluorescent Pseudomonas (viable count ≥300 billion / g): Shandong Huimin Zhonglian Biotechnology Co., Ltd.;

[0042] Bacillus subtilis (live count ≥ 50 billion / g): Zhucheng Huikefeng Biotechnology Co., Ltd.;

[0043] Paecilomyces lilacinus (viable count ≥10 billion / g): Guangxi Nongbao Bioengineering Co., Ltd.;

[0044] Streptomyces freundii (viable count ≥ 200 million / g): Shaanxi Xinfengkai Crop Protection Co., Ltd.;

[0045] Bacillus megaterium (live count ≥ 10 billion / g): Guangxi Kanglv Biotechnology Co., Ltd.

[0046] 3. Test crops:

[0047] Tobacco (Yunyan 87): Pingtang County Branch of Qiannan Prefecture Tobacco Company.

[0048] III. Detection Methods

[0049] 1. Detection of amide herbicides in soil samples

[0050] Accurately weigh 10 ± 0.01 g of soil sample into a 50 mL centrifuge tube, add 10 mL of 0.5% acetic acid-acetonitrile solution, and vortex for 10 min (2500 rpm). Then add 1 g of sodium chloride and 1 g of anhydrous magnesium sulfate, vortex for 10 min (2500 rpm), and centrifuge at 5000 rpm for 6 min. Transfer 1 mL of the supernatant to a container containing 50 mg C. 18 The sample was vortexed for 1 min in centrifuge tubes. It was then filtered through a 0.22 μm organic filter membrane and analyzed by HPLC-HRMS under the conditions shown in Table 1 below.

[0051] Table 1. Optimal detection conditions for three amide herbicides by HPLC-HRMS.

[0052]

[0053]

[0054] 2. Soil microbial community detection

[0055] All PCR mixtures were added to 15 μL of Phusion High-Fidelity PCR Master Mix, 0.2 μm primers, and 10 ng of genomic DNA template. The mixture was first denatured at 98 °C for 1 min, followed by 30 cycles at 98 °C (10 s), 50 °C (30 s), and 72 °C (30 s), and finally held at 72 °C for 5 min. The V4-V5 region of the bacterial 16S rRNA gene was amplified using primers 515F (GTGCCAGCMGCCGCGGTAA) and 907R (CCGTCAATTCCTTTGAGT TT). Sequencing was performed on the Novaseq-PE250 platform, and species annotation was performed using QIIME2 software. Representative sequences for each OTU were annotated using the Silva database.

[0056] IV. Field Trials

[0057] A. Test Plan

[0058] The experiment was conducted in Kedu Town, Pingtang County, from April to October 2024. The experimental plots were relatively flat, with convenient irrigation and drainage, and the soil was loose and moderately fertile. Application of herbicides was carried out in April 2024, with acetochlor (2700 g ai / ha), bensulfuron-methyl (1500 g ai / ha), and metolachlor (3240 g ai / ha) mixed and applied evenly to the ridge surface at twice the recommended field dosage. Two control groups were set up: a blank control group (no herbicide application, planted with normal tobacco plants) and a herbicide control group (herbicide applied only without the addition of microbial agents, resulting in herbicide-damaged tobacco plants). The tobacco seedlings were transplanted 20 days after application.

[0059] Ten days after transplanting tobacco seedlings, root irrigation experiments were conducted using either direct dilution of the microbial agent with water or fermentation broth of the microbial agent. The direct dilution root irrigation experiment included six remediation treatments: 500-fold dilution of Bacillus subtilis, 700-fold dilution of Bacillus megaterium, 300-fold dilution of Pseudomonas fluorescens, 800-fold dilution of Streptomyces freundii, 500-fold dilution of Trichoderma longifolia, and 500-fold dilution of Paecilomyces lilacinus. Each treatment was administered at a dose of 500 mL per plant.

[0060] Similarly, the root irrigation experiment with the microbial agent fermentation broth included six treatment groups. The fermentation process involved mixing the microbial agent, fermentable carbon source, and water at a mass ratio of 1:5:100, fermenting in the dark for 7 days, and then diluting before use. All the above-mentioned microbial agent fermentation broths were diluted 50 times and then applied to the roots at a dosage of 500 mL per plant.

[0061] The tobacco plants were irrigated with a second and third application of the foliar spray at 30 and 50 days after transplanting. Each treatment group and the control group had three replicate plots, each plot measuring 10 m². 2 Field management and pest and disease control strictly adhere to local standards for high-quality tobacco production.

[0062] Tobacco plant height, stem circumference, maximum leaf length, and maximum leaf width were measured at 30 and 70 days after transplanting. Soil samples were collected at 2 hours, 28, 42, and 70 days after pesticide application, using a quartering method to collect samples from the 0-15 cm topsoil layer. After aliquoting, one sample was stored at -20℃ for residue detection, and the other was frozen at -80℃ for microbial community analysis.

[0063] B. Test Results

[0064] (I) Agronomic traits of tobacco

[0065] 1. Agronomic traits of tobacco plants 30 days after transplanting: treatment with microbial fermentation broth and direct root irrigation with diluted microbial agent.

[0066] Thirty days after transplanting, the maximum leaf length, maximum leaf width, plant height, and stem circumference of the tobacco were measured. Figure 1Data showed that the growth indicators of the herbicide control treatment were all significantly lower than those of the blank control. Among them, the maximum leaf length, maximum leaf width, plant height, and stem circumference were only 52.38%, 30.62%, 19.81%, and 23.14% of those of the blank control, respectively, indicating that the combined residues of the three amide herbicides in the soil had a significant inhibitory effect on tobacco seedling growth.

[0067] The comparison of tobacco agronomic traits at 30 days after transplanting between treatments with fermentation broth of six microbial agents and treatments with direct application of the agents diluted with water is shown in the figure. Figure 2 As shown in the figure, the agronomic traits of tobacco plants treated with the fermentation broths of the six microbial agents were generally better than those treated with direct application of the agents diluted with water. Specifically, the fermentation broth treatments of *Bacillus subtilis*, *Pseudomonas fluorescens*, *Streptomyces freundii*, and *Paecilomyces lilacinus* showed significant differences compared to the direct application of the agents in all agronomic traits. The *Trichoderma longicornis* fermentation broth treatment showed a significant improvement in maximum leaf length compared to the direct application of the agents, while leaf width, plant height, and stem circumference showed no statistically significant differences. The *Bacillus megaterium* fermentation broth treatment showed no significant differences compared to the direct application of the agents in any of the four indicators.

[0068] In terms of maximum leaf length, the fermentation broth treatment of Paecilomyces lilacinus and Streptomyces freundii showed the best improvement, increasing by 64.62% and 63.10% respectively compared to the treatment of direct application of the inoculant diluted with water. Pseudomonas fluorescens, Trichoderma longifolia, Bacillus subtilis and Bacillus megaterium increased by 54.68%, 36.60%, 30.20% and 27.94% respectively.

[0069] In terms of maximum leaf width, the fermentation broth treatments of *Paecilomyces lilacinus* and *Streptomyces freundii* showed the best improvement, increasing the width by 119.77% and 113.45% respectively compared to the direct application of the inoculant diluted with water. *Pseudomonas fluorescens*, *Bacillus subtilis*, and *Bacillus megaterium* showed increases of 52.54%, 35.66%, and 31.80%, respectively. However, the maximum leaf width treatment with *Trichoderma longicornis* fermentation broth only increased by 11.70% compared to the direct application of the inoculant diluted with water.

[0070] In terms of plant height, the *Streptomyces freundii* fermentation broth treatment showed the best improvement, increasing height by 261.54% compared to the direct application of the inoculant diluted with water. *Paecilomyces lilacinus*, *Pseudomonas fluorescens*, and *Bacillus subtilis* treatments showed increases of 159.52%, 153.62%, and 143.90%, respectively. In contrast, the plant height increases with *Bacillus megaterium* and *Trichoderma longicornis* fermentation broth treatments were only 37.42% and 16.44% higher, respectively, than the direct application of the inoculant diluted with water treatment.

[0071] In terms of stem circumference, the Bacillus subtilis fermentation broth treatment showed the best improvement, increasing the circumference by 105.88% compared to the direct application of the inoculant diluted with water. Pseudomonas fluorescens, Paecilomyces lilacinus, and Streptomyces freundii treatments showed increases of 86.67%, 65.16%, and 63.24%, respectively. However, the plant height treated with Bacillus megaterium and Trichoderma longifolia fermentation broth only increased by 31.78% and 20%, respectively, compared to the direct application of the inoculant diluted with water treatment.

[0072] The agronomic traits of tobacco treated with fermentation broths of six microbial agents at 30 days after transplanting are shown in the figure. Figure 3 As can be seen from the figure, the treatment effect of *Streptomyces freundii* fermentation broth was the best, and its various agronomic traits were significantly better than those of other inoculant fermentation broths.

[0073] The results show that, 30 days after transplanting tobacco seedlings, the treatment with *Streptomyces freundii* fermentation broth was the most effective, followed by *Pseudomonas fluorescens* and *Paecilomyces lilacinus*.

[0074] 2. Agronomic traits of tobacco treated with fermentation broths of six microbial agents 70 days after transplanting

[0075] Plant height, stem circumference, maximum leaf length, and leaf width of tobacco plants treated with herbicides and those treated with a blank control were measured 70 days after transplanting. Figure 4 Compared with the blank control, the four growth indicators of the herbicide control treatment were significantly reduced: the maximum leaf length, leaf width, plant height and stem circumference were only 47.79%, 53.62%, 37.38% and 36.21% of the blank control, respectively, indicating that the residues of the three amide herbicides in the soil had a significant inhibitory effect on tobacco seedling growth.

[0076] from Figure 5 As can be seen, there were significant differences in maximum leaf length among the six microbial inoculant fermentation broth treatments compared to the herbicide control. Compared to the herbicide control, the *Streptomyces freundii* and *Paecilomyces lilacinus* fermentation broth treatments showed the best improvement, increasing by 72.68% and 70.24% respectively. *Pseudomonas fluorescens*, *Trichoderma longifolia*, *Bacillus megaterium*, and *Bacillus subtilis* treatments increased by 57.5%, 54.15%, 40.98%, and 40.24% respectively.

[0077] from Figure 6 As can be seen, in terms of maximum leaf width, *Bacillus megaterium* did not reach a significant level compared to the herbicide control, while the fermentation broth treatments of the other five inoculants all showed significant differences. Compared to the herbicide control, the fermentation broth treatments of *Paecilomyces lilacinus* and *Streptomyces freundii* showed the best improvement, increasing by 90.96% and 88.55% respectively, while *Pseudomonas fluorescens*, *Bacillus subtilis*, and *Trichoderma longicornis* increased by 59.64%, 52.17%, and 36.14% respectively. However, the fermentation broth treatment of *Bacillus megaterium* only increased by 23.49% compared to the herbicide control.

[0078] from Figure 7 As can be seen, in terms of plant height, all six microbial inoculant fermentation broth treatments showed significant differences compared to the herbicide control. Compared to the herbicide control, the fermentation broth treatments of *Paecilomyces lilacinus*, *Streptomyces freundii*, and *Trichoderma longicornis* showed the best improvement, increasing height by 166.89%, 146.49%, and 143.64%, respectively. *Bacillus subtilis*, *Bacillus megaterium*, and *Pseudomonas fluorescens* showed increases of 100%, 93.86%, and 89.04%, respectively.

[0079] from Figure 8 As can be seen, in terms of stem circumference, all six microbial inoculant fermentation broth treatments showed significant differences compared to the herbicide control, with the *Streptomyces freundii* fermentation broth treatment showing the most significant difference. Compared to the herbicide control, the *Streptomyces freundii* and *Paecilomyces lilacinus* fermentation broth treatments showed the best improvement, increasing by 173.81% and 169.05% respectively. *Trichoderma longifolia*, *Bacillus subtilis*, *Pseudomonas fluorescens*, and *Bacillus megaterium* increased by 139.52%, 130.95%, 88.10%, and 83.33% respectively.

[0080] Comprehensive analysis showed that the fermentation broth treatment with *Streptomyces freundii* and *Paecilomyces lilacinus* was the most effective in promoting leaf development and stem growth, followed by the fermentation broth treatment with *Pseudomonas fluorescens*.

[0081] Based on agronomic trait data of tobacco seedlings 30 and 70 days after transplanting, fermentation broths of Paecilomyces lilacinus and Streptomyces freundii can effectively alleviate the inhibitory effect of amide herbicides on tobacco growth.

[0082] (II) Degradation effect of amide herbicides

[0083] 1. Effects of fermentation broth treatments of various microbial agents on the degradation of acetochlor in tobacco field soil.

[0084] As shown in Table 2, the acetochlor residue levels in all treatment groups were basically consistent 2 hours after application: 6.95 mg / kg for the herbicide control group, and 7.72, 7.01, 7.52, 6.67, 6.27, and 6.45 mg / kg for the fermentation broth treatments of *Paecilomyces lilacinus*, *Streptomyces freundii*, *Trichoderma longifolia*, *Bacillus megaterium*, *Bacillus subtilis*, and *Pseudomonas fluorescens*, respectively. Subsequent tests (28 days, 42 days, and 70 days) showed that the acetochlor residue levels in all fermentation broth treatments were significantly lower than those in the herbicide control.

[0085] Twenty-eight days after application (eight days after transplanting tobacco seedlings), the residue of acetochlor in the herbicide control group was 1.87 mg / kg. In contrast, the residues in samples treated with fermentation broths of *Streptomyces freundii*, *Pseudomonas fluorescens*, and *Paecilomyces lilacinus* decreased to 0.89, 1.17, and 1.22 mg / kg, respectively. The acetochlor residues in the herbicide control group were 2.1 times, 1.6 times, and 1.5 times higher than those treated with these three microbial fermentation broths. Furthermore, the residues in samples treated with fermentation broths of *Bacillus megaterium*, *Bacillus subtilis*, and *Trichoderma longifolia* were 1.32, 1.31, and 1.53 mg / kg, respectively. The acetochlor residues in the herbicide control group were 1.4 times, 1.4 times, and 1.2 times higher than those treated with these three microbial fermentation broths.

[0086] Forty-two days after application (22 days after transplanting tobacco seedlings), the residue of acetochlor in the herbicide control group was 1.30 mg / kg. However, in samples treated with fermentation broths of *Streptomyces freundii*, *Pseudomonas fluorescens*, and *Paecilomyces lilacinus*, the residue levels decreased to 0.41, 0.61, and 0.65 mg / kg, respectively. The acetochlor residue levels in the herbicide control group were 3.2 times, 2.1 times, and 2.0 times higher than those treated with these three microbial fermentation broths. Furthermore, the residue levels in samples treated with fermentation broths of *Bacillus megaterium*, *Trichoderma longifolia*, and *Bacillus subtilis* were 0.69, 0.74, and 0.91 mg / kg, respectively. The acetochlor residue levels in the herbicide control group were 1.9 times, 1.8 times, and 1.4 times higher than those treated with these three microbial fermentation broths.

[0087] At 70 days after application (50 days after transplanting tobacco seedlings), the acetochlor residue in the herbicide control group was 0.81 mg / kg. However, in samples treated with fermentation broths of *Streptomyces freundii*, *Bacillus megaterium*, *Paecilomyces lilacinus*, and *Pseudomonas fluorescens*, the residue levels decreased to 0.13, 0.18, 0.28, and 0.29 mg / kg, respectively. The acetochlor residue in the herbicide control group was 6.2 times, 4.5 times, 2.9 times, and 2.8 times higher than that treated with these four microbial fermentation broths. Furthermore, the residue levels in samples treated with fermentation broths of *Trichoderma longifolia* and *Bacillus subtilis* were 0.54 and 0.65 mg / kg, respectively. The acetochlor residue in the herbicide control group was 1.5 times and 1.2 times higher than that treated with these two microbial fermentation broths.

[0088] In terms of degradation rate, the degradation rate of acetochlor in all microbial inoculant fermentation broth treatments was higher than that of the herbicide control. At 28 days after application (8 days after transplanting tobacco seedlings), the degradation rates of all six microbial inoculant fermentation broth treatments were significantly different from the herbicide control. The *Streptomyces freundii* fermentation broth treatment had the highest degradation rate at 87.37%, followed by *Paecilomyces lilacinus* at 84.24%. At 42 days after application (22 days after transplanting tobacco seedlings), the degradation rates of all six microbial inoculant fermentation broth treatments were also significantly different from the herbicide control. The *Streptomyces freundii* fermentation broth treatment had the highest degradation rate at 94.17%, followed by *Paecilomyces lilacinus* at 91.58%.

[0089] At 70 days after application (50 days after transplanting tobacco seedlings), except for the Bacillus subtilis treatment, the degradation rates of the other five treatments were significantly different from the herbicide control. The Streptomyces freundii fermentation broth treatment had the highest degradation rate at 98.16%, followed by Bacillus megaterium at 97.26%.

[0090] In summary, *Streptomyces freundii* fermentation broth treatment showed the best degradation effect on acetochlor, followed by *Paecilomyces lilacinus* and *Pseudomonas fluorescens*. All three significantly improved the degradation efficiency of acetochlor and greatly reduced its residue.

[0091] Table 2. Residual amounts and degradation rates of acetochlor in fermentation broth treated with different microbial agents.

[0092]

[0093]

[0094] 2. Effects of fermentation broth treatments of various microbial agents on the degradation of metolachlor in tobacco field soil.

[0095] As shown in Table 3, the residual levels of metolachlor in each treatment group were basically consistent 2 hours after application: 11.54 mg / kg for the herbicide control group, and 11.44, 10.99, 10.83, 10.14, 11.86, and 9.71 mg / kg for the fermentation broth treatments of *Paecilomyces lilacinus*, *Streptomyces freundii*, *Trichoderma longifolia*, *Bacillus megaterium*, *Bacillus subtilis*, and *Pseudomonas fluorescens*, respectively. Subsequent tests (28, 42, and 70 days) showed that the residual levels of metolachlor in all fermentation broth treatments were significantly lower than those in the herbicide control. At 28 days after application (8 days after transplanting tobacco seedlings), the residual level of metolachlor in the herbicide control group was 3.44 mg / kg. In samples treated with fermentation broths of *Paecilomyces lilacinus*, *Streptomyces freundii*, *Pseudomonas fluorescens*, and *Trichoderma longicornis*, the residue levels decreased to 1.16, 1.31, 1.47, and 1.56 mg / kg, respectively. The residue levels of metolachlor in the herbicide control group were 3.0, 2.6, 2.3, and 2.2 times higher than those treated with these four microbial agents. Furthermore, the residue levels of *Bacillus megaterium* and *Bacillus subtilis* fermentation broths were 2.10 and 2.58 mg / kg, respectively. The residue levels of metolachlor in the herbicide control group were 1.6 and 1.3 times higher than those treated with these two microbial agents.

[0096] Forty-two days after application (22 days after transplanting tobacco seedlings), the residue of metolachlor in the herbicide control group was 2.45 mg / kg. In samples treated with fermentation broths of *Streptomyces freundii*, *Paecilomyces lilacinus*, and *Pseudomonas fluorescens*, the residue levels decreased to 0.81, 0.89, and 0.95 mg / kg, respectively. The metolachlor residue levels in the herbicide control group were 3.0 times, 2.8 times, and 2.6 times higher than those treated with these three microbial fermentation broths. Furthermore, the residue levels in samples treated with fermentation broths of *Bacillus megaterium*, *Trichoderma longifolia*, and *Bacillus subtilis* were 1.10, 1.19, and 1.97 mg / kg, respectively. The metolachlor residue levels in the herbicide control group were 2.2 times, 2.1 times, and 1.2 times higher than those treated with these three microbial fermentation broths.

[0097] At 70 days after application (50 days after transplanting tobacco seedlings), the residue of metolachlor in the herbicide control group was 1.52 mg / kg. However, in samples treated with fermentation broths of *Pseudomonas fluorescens*, *Streptomyces flexneri*, *Bacillus megaterium*, and *Paecilomyces lilacinus*, the residue levels decreased to 0.19, 0.36, 0.45, and 0.49 mg / kg, respectively. The metolachlor residue in the herbicide control group was 7.9 times, 4.2 times, 3.4 times, and 3.1 times higher than that treated with these four microbial fermentation broths. Furthermore, the residue levels in samples treated with fermentation broths of *Trichoderma longifolia* and *Bacillus subtilis* were 0.59 and 0.65 mg / kg, respectively. The metolachlor residue in the herbicide control group was 2.6 times and 2.3 times higher than that treated with these two microbial fermentation broths.

[0098] In terms of degradation rate, the degradation rate of isopropylate in all microbial inoculant fermentation broth treatments was higher than that of the herbicide control. At 28 days after application (8 days after transplanting), the degradation rates of all six microbial inoculant fermentation broth treatments were significantly different from the herbicide control. The *Paecilomyces lilacinus* fermentation broth treatment had the highest degradation rate at 89.83%, followed by *Streptomyces freundii* at 88.06%. At 42 days after application (22 days after transplanting), the degradation rates of all six microbial inoculant fermentation broth treatments were also significantly different from the herbicide control. The *Streptomyces freundii* fermentation broth treatment had the highest degradation rate at 92.67%, followed by *Paecilomyces lilacinus* at 92.21%. At 70 days after application (50 days after transplanting), the degradation rates of all six microbial inoculant fermentation broth treatments were also significantly different from the herbicide control. The *Pseudomonas fluorescens* fermentation broth treatment had the highest degradation rate at 98.09%, followed by *Streptomyces freundii* at 96.46%.

[0099] In summary, the fermentation broth treatment of *Pseudomonas fluorescens* showed the best degradation effect on isopropylate, followed by *Paecilomyces lilacinus* and *Streptomyces freundii*. All three significantly improved the degradation efficiency and greatly reduced its residue.

[0100] Table 3. Residue and degradation rate of isopropyl chlorpyrifos in fermentation broth treated with different microbial agents.

[0101]

[0102] 3. Effects of fermentation broth treatments of various microbial agents on the degradation of bensulfuron-methyl in tobacco field soil.

[0103] As shown in Table 3, the residual levels of benzylpyr in each treatment group were basically consistent 2 hours after application: 6.59 mg / kg for the herbicide control group, and 5.85, 6.80, 6.15, 6.73, 6.06, and 6.32 mg / kg for the fermentation broth treatments of *Paecilomyces lilacinus*, *Streptomyces freundii*, *Trichoderma longifolia*, *Bacillus megaterium*, *Bacillus subtilis*, and *Pseudomonas fluorescens*, respectively. Subsequent tests (28, 42, and 70 days) showed that the residual levels of benzylpyr in all microbial agent treatments were significantly lower than those in the herbicide control. At 28 days after application (8 days after transplanting tobacco seedlings), the residual level of benzylpyr in the herbicide control group was 1.34 mg / kg. In samples treated with fermentation broths of *Paecilomyces lilacinus*, *Streptomyces freundii*, and *Pseudomonas fluorescens*, the residue levels decreased to 0.61, 0.80, and 0.84 mg / kg, respectively. The residue levels of benzyl-methyl in the herbicide control group were 2.1 times, 1.7 times, and 1.6 times higher than those treated with these three microbial fermentation broths, respectively. Furthermore, the residue levels of *Trichoderma longifolia*, *Bacillus megaterium*, and *Bacillus subtilis* fermentation broths were 0.91, 0.92, and 1.02 mg / kg, respectively. The residue levels of benzyl-methyl in the herbicide control group were 1.5 times, 1.5 times, and 1.3 times higher than those treated with these three microbial fermentation broths, respectively. At 42 days after application (22 days after transplanting tobacco seedlings), the residue level of benzyl-methyl in the herbicide control group was 0.76 mg / kg. In samples treated with fermentation broths of *Streptomyces freundii*, *Bacillus subtilis*, and *Paecilomyces lilacinus*, the residue levels decreased to 0.12, 0.17, and 0.21 mg / kg, respectively. The residue levels of benzyl-methyl in the herbicide control group were 6.3 times, 4.5 times, and 3.6 times higher than those treated with these three microbial fermentation broths, respectively. Furthermore, the residue levels of *Pseudomonas fluorescens*, *Trichoderma longifolia*, and *Bacillus megaterium* fermentation broths were 0.26, 0.27, and 0.29 mg / kg, respectively. The residue levels of benzyl-methyl in the herbicide control group were 2.9 times, 2.8 times, and 2.6 times higher than those treated with these three microbial fermentation broths, respectively. At 70 days after application (50 days after transplanting tobacco seedlings), the residue level of benzyl-methyl in the herbicide control group was 0.29 mg / kg. In samples treated with fermentation broths of *Streptomyces freundii*, *Paecilomyces lilacinus*, and *Pseudomonas fluorescens*, the residue levels decreased to 0.05, 0.07, and 0.09 mg / kg, respectively. The residue levels of benzyl-methyl in the herbicide control group were 5.8 times, 4.1 times, and 3.2 times higher than those treated with these three microbial agents. Furthermore, the residue levels of fermentation broths treated with *Bacillus megaterium*, *Bacillus subtilis*, and *Trichoderma longifolia* were 0.10, 0.13, and 0.14 mg / kg, respectively. The residue levels of benzyl-methyl in the herbicide control group were 2.9 times, 2.2 times, and 2.1 times higher than those treated with these three microbial agents.

[0104] In terms of degradation rate, the degradation rate of benzyl oxychloride in all microbial fermentation broth treatments was higher than that of the herbicide control. At 28 days after application (8 days after transplanting), except for the Bacillus subtilis treatment, the degradation rates of the other five treatments were significantly different from the herbicide control. The Paecilomyces lilacinus fermentation broth treatment had the highest degradation rate at 89.53%, followed by Streptomyces freundii at 88.18%. At 42 days after application (22 days after transplanting), the degradation rates of all six microbial fermentation broth treatments were significantly different from the herbicide control. The Streptomyces freundii fermentation broth treatment had the highest degradation rate at 98.18%, followed by Bacillus subtilis at 97.12%. At 70 days after application (50 days after transplanting), the degradation rates of all six microbial fermentation broth treatments were significantly different from the herbicide control. The Streptomyces freundii fermentation broth treatment had the highest degradation rate at 99.34%, followed by Pseudomonas fluorescens at 98.91%. In summary, the fermentation broth treatment of *Streptomyces freundii* showed the best degradation effect on benzyl sulfadiazine, followed by *Paecilomyces lilacinus* and *Pseudomonas fluorescens*. All three significantly improved the degradation efficiency and greatly reduced the residue.

[0105] Table 4. Residue and degradation rate of bensulfuron-methyl in fermentation broth treated with different microbial agents.

[0106]

[0107]

[0108] (III) Soil microbial diversity

[0109] By analyzing the microbial community structure and diversity, this study comprehensively evaluated the impact of microbial inoculant fermentation broth on mitigating tobacco herbicide damage caused by compound residues of amide herbicides 70 days after application (50 days after transplanting). Figure 9 The Venn diagram showed a total of 427 bacterial OTUs across all treatment groups, indicating that these OTUs were not significantly affected by the microbial agents. The herbicide control group (CKY) had 214 unique OTUs, while the Paecilomyces lilacinus (PL), Bacillus subtilis (BS), and Streptomyces freundii (SF) fermentation broth treatment groups contained 523, 305, and 274 unique OTUs, respectively. Studies have shown that unique genera within specific microbial communities can significantly influence the migration, transformation, and degradation processes of pollutants.

[60] Compared with the herbicide control group, the number of specific bacterial genera in the fermentation broth treatment groups of the three microbial agents was significantly increased, and these genera may play a key role in the degradation of amide herbicides.

[0110] Figure 10The study presented the composition of the top 10 dominant bacterial communities at the phylum level. The top five most abundant phyla were: Chloroflexi (18.11%-51.61%), Proteobacteria (11.29%-38.75%), Actinobacteriota (15.79%-27.80%), Gemmatimonadota (0.44%-10.65%), and Acidobacteriota (3.90%-8.45%). The relative abundance of Chloroflexi was lower in all microbial inoculant fermentation broth treatments than in the herbicide control group, with a reduction of 65.38% in the *Streptomyces freundii* fermentation broth treatment and 59% in the *Paecilomyces lilacinus* fermentation broth treatment. Studies have shown that the filamentous morphology of *Chlorophyta* makes it easy for them to form biofilm structures in soil micro-aggregates. These physical barriers may prevent degrading bacteria such as *Nocardia* and *Brevibacterium* from contacting pollutants, thereby inhibiting degradation efficiency. [61 , 62] The abundance of Bacillus spp. in the Paecilomyces lilacinus fermentation broth treatment group was higher than that in other treatment groups, suggesting that this genus may play a key role in the degradation of amide herbicides.

[0111] Figure 11 The top 10 dominant bacterial communities at the genus level were shown. The top five most abundant genera were: Chujaibacter (3.49%-25.45%), Mizugakiibacter (0.40%-11.29%), Rhodanobacter (0.02%-5.67%), Acidothermus (1.03%-4.25%), and Acidipila Silvibacterium (0.50%-3.43%). The abundance of Chujaibacter in the *Streptomyces freundii* fermentation broth treatment group was significantly higher than in other treatment groups, while the abundance of Rhodanobacter in the *Paecilomyces lilacinus* fermentation broth treatment group was significantly higher. This may be an important reason for the higher degradation efficiency of amide herbicides in both groups.

[0112] Compared with the herbicide control group, treatment with *Paecilomyces lilacinus* fermentation broth significantly increased the relative abundance of species such as *Gemmatimonas* sp., *Streptomyces lincolnensis*, and *Rhodanobacter* sp. Figure 12 Some studies suggest that Gemmatimonas sp. may indirectly promote plant growth by secreting growth-promoting factors or establishing symbiotic relationships with plant roots when participating in the decomposition of organic matter and the carbon cycle. [63,64]Meanwhile, Streptomyces_lincolnensis and Rhodanobacter_sp participate in nitrogen and phosphorus cycling in the rhizosphere soil by carrying specific functional genes, thereby enhancing plant nutrient absorption and growth.

[65] The functional characteristics of these microorganisms may be an important mechanism by which they alleviate crop pesticide damage.

[0113] Table 5 shows that there were significant differences in soil microbial community diversity indices among the six fermentation broth treatments. The Shannon and Simpson indices for the herbicide control group were 6.48 and 0.945, respectively. Treatments with *Paecilomyces lilacinus* and *Streptomyces freundii* fermentation broth significantly increased these two indices: the Shannon indices reached 7.10 and 6.99, respectively, representing increases of 9.57% and 7.87% compared to the control group; the Simpson indices reached 0.976 and 0.974, respectively, representing increases of 3.28% and 3.07% compared to the control group. Regarding species richness, the Ace and Chao indices for the herbicide control group were 1190.50 and 1081.13, respectively. The Ace index of the *Paecilomyces lilacinus* fermentation broth treatment was 1759.15, an increase of 47.89% compared to the control group, and the Chao index was 1544.83, an increase of 42.77%. The Ace index of the *Streptomyces freundii* fermentation broth treatment was 1752.56, an increase of 47.21% compared to the control group, and the Chao index was 1409.08, an increase of 30.33%. These data indicate that the fermentation broth treatments of both microbial agents significantly improved the species richness and diversity of the soil microbial community, which may be related to their ability to mitigate the toxic effects of amide herbicides residues on tobacco plants.

[0114] Table 5. Bacterial microbial community diversity index under different microbial inoculant fermentation broth treatments

[0115]

[0116]

[0117] V. Pot Experiment

[0118] A. Test Plan

[0119] The soil samples were taken from Huaxi District, Guiyang City, Guizhou Province, at a depth of 0-15 cm. After being air-dried indoors, impurities were removed, and the soil samples were ground and sieved through a 2 mm sieve for later use.

[0120] This study investigated the degradation effects of fermentation broths from three microbial inoculants on residues of amide herbicides (acetochlor, metolachlor, and bensulfuron-methyl) in compoundly contaminated soil using an indoor pot simulation method. Four treatment groups were set up: a herbicide control group, a *Pseudomonas fluorescens* group, a *Paecilomyces lilacinus* group, and a *Streptomyces freundii* group, with five replicates per group, totaling 20 pots. Three herbicides (at field recommended concentrations) were added to the soil using an artificial contamination method to create a compound contamination environment. The fermentation broths were applied on the same day as the herbicides were applied. The fermentation process involved mixing the inoculant, fermentation-type carbon source, and water at a mass ratio of 1:5:100, fermenting in the dark for 7 days, and then diluting before use. All fermentation broths were diluted 50 times and administered as a root drench at a dose of 500 mL per plant. Sampling was conducted 2 hours after application and at 1, 3, 5, 7, 10, 14, 21, 28, 35, and 42 days after application. Samples were stored at -20°C for residual detection.

[0121] B. Test Results

[0122] 1. Study on the residual decomposition of acetochlor in soil by microbial inoculant fermentation broth

[0123] like Figure 13 As shown, the acetochlor residue levels in all treatment groups were essentially the same within 2 hours after application. From day 1 after application, the residue levels in all microbial inoculant fermentation broth treatments were lower than those in the herbicide control group. The *Streptomyces freundii* fermentation broth treatment group showed the fastest degradation rate in the first 7 days, followed by a slower degradation rate. By day 42 after application, the acetochlor residue level in the herbicide control group was 0.16 mg / kg, while the *Streptomyces freundii*, *Pseudomonas fluorescens*, and *Paecilomyces lilacinus* fermentation broth treatment groups decreased to 0.03, 0.05, and 0.08 mg / kg, respectively. At this point, the residue levels in the three microbial inoculant treatments were equivalent to 18.75%, 31.25%, and 50.00% of those in the herbicide control group, respectively.

[0124] Table 6 shows the degradation of acetochlor under different treatment conditions. The half-life of acetochlor in the herbicide control group was 12.60 days, while the half-life of the microbial fermentation broth treatment group was significantly shorter. This indicates that the fermentation broths of the three microbial agents can effectively promote the degradation of acetochlor in the soil. Specifically, the half-life of the *Streptomyces freundii* fermentation broth treatment group was the shortest, at 7.70 days; followed by the *Pseudomonas fluorescens* fermentation broth treatment group with a half-life of 8.66 days, and the half-life of the *Paecilomyces lilacinus* fermentation broth treatment group was 10.04 days. As shown in Table 6-2, the degradation rates of acetochlor in the *Streptomyces freundii* and *Pseudomonas fluorescens* fermentation broth treatment groups were significantly different from those in the herbicide control group at all detection time points, while the *Paecilomyces lilacinus* treatment group showed no significant difference only on days 5 and 10 after application.

[0125] Comprehensive analysis shows that the fermentation broths of the three microbial agents can accelerate the degradation process of acetochlor, and their effects are in the following order: Streptomyces freundii > Pseudomonas fluorescens > Paecilomyces lilacinus.

[0126] Table 6. Degradation rate, degradation kinetic equation, and degradation half-life of acetochlor in soil under different treatments.

[0127]

[0128] 2. Study on the residual decomposition of metolachlor in soil by microbial inoculant fermentation broth

[0129] like Figure 14 As shown, the residue levels of isopropylate in all treatment groups were basically the same within 2 hours after application. From day 1 after application, the residue levels in the *Streptomyces freundii* and *Pseudomonas fluorescens* fermentation broth treatment groups were lower than those in the herbicide control group, while the *Paecilomyces lilacinus* fermentation broth treatment group also showed a decreasing trend from day 3. By day 42 after application, the residue level in the herbicide control group was 0.35 mg / kg, while the residue levels in the *Streptomyces freundii*, *Pseudomonas fluorescens*, and *Paecilomyces lilacinus* fermentation broth treatment groups decreased to 0.12, 0.08, and 0.17 mg / kg, respectively, equivalent to 34.29%, 22.86%, and 48.57% of the herbicide control group residue levels.

[0130] Table 7 shows the degradation of metolachlor under different treatment conditions. The half-life of metolachlor in the herbicide control group was 16.12 days, while the half-life of the microbial fermentation broth treatment groups was significantly shorter, indicating that the three microbial fermentation broths can effectively promote the degradation of metolachlor in the soil. Specifically, the half-life of the *Pseudomonas fluorescens* fermentation broth treatment group was the shortest (8.88 days), followed by the *Streptomyces freundii* fermentation broth treatment group (9.90 days), and the half-life of the *Paecilomyces lilacinus* fermentation broth treatment group was 11.36 days. As shown in Table 6-3, the degradation rate of metolachlor in the three microbial fermentation broth treatment groups at each detection time point was significantly different from that of the herbicide control group.

[0131] Comprehensive analysis shows that the fermentation broths of the three microbial agents can accelerate the degradation process of metolachlor, and their effects are in the following order: Pseudomonas fluorescens > Streptomyces freundii > Paecilomyces lilacinus.

[0132] Table 7. Degradation kinetics and half-life of metolachlor in soil under different treatments.

[0133]

[0134] 3. Study on the residual decomposition of benzyl oxychloride in soil by microbial inoculant fermentation broth

[0135] like Figure 15As shown, the residue levels of bensulfuron-methyl in each treatment group were basically the same within 2 hours after application. From the first day after application, the residue levels in each microbial fermentation broth treatment were lower than those in the herbicide control group. The *Streptomyces freundii* fermentation broth treatment group showed the fastest degradation rate in the first 3 days, followed by a slower degradation rate. By day 42 after application, the residue level of bensulfuron-methyl in the herbicide control group was 0.20 mg / kg, while the residue levels in the *Streptomyces freundii*, *Pseudomonas fluorescens*, and *Paecilomyces lilacinus* fermentation broth treatment groups decreased to 0.07, 0.12, and 0.11 mg / kg, respectively. At this point, the residue levels in the three microbial fermentation broth treatments were equivalent to 35%, 60%, and 55% of those in the herbicide control group, respectively.

[0136] Table 8 shows the degradation of benzylpyr under different treatment conditions. The half-life of benzylpyr in the herbicide control group was 11.36 days, while the half-life of the inoculant fermentation broth treatment groups was significantly shorter, indicating that the three inoculant fermentation broths can effectively promote the degradation of benzylpyr in the soil. Specifically, the half-life of the *Streptomyces freundii* fermentation broth treatment group was the shortest (8.35 days), followed by the *Pseudomonas fluorescens* and *Paecilomyces lilacinus* fermentation broth treatment groups, both with a half-life of 9.76 days. As shown in Table 6-4, the degradation rate of benzylpyr in the three inoculant fermentation broth treatment groups at each detection time point was significantly different from that of the herbicide control group.

[0137] Comprehensive analysis shows that the fermentation broths of the three microbial agents can accelerate the degradation process of benzyl sulfadiazine, and their effects are in the following order: Streptomyces freundii > Pseudomonas fluorescens > Paecilomyces lilacinus.

[0138] Table 8. Degradation kinetics and half-life of bensulfuron-methyl in soil under different treatments

[0139]

[0140]

[0141] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. Application of *Streptomyces freundii* fermentation broth in any one or more of the following: (1) Promotes the degradation of amide herbicides in the soil; (2) Improve the community structure of soil microorganisms, among which, Decrease the abundance of Pleuroscylformes, and increase the abundance of Bacillus, Proteobacteria, and functional genera.

2. The application according to claim 1, characterized in that, The Streptomyces freundii fermentation broth is made by mixing Streptomyces freundii agent, fermentation-type carbon source and water, fermenting in the dark and then diluting before use.

3. The application according to claim 2, characterized in that, The Streptomyces freundii agent, fermentable carbon source, and water are mixed in a mass ratio of 1:5:

100.

4. The application according to claim 1, characterized in that, The amide herbicide is any one or more of acetochlor, metolachlor, and bensulfuron-methyl.

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