A method for synergistic enhancement of phytoremediation of heavy metal contaminated soil using nitrogen-sulfur doped carbon quantum dots and plant endophytic bacteria.

Abstract

This invention discloses a method for synergistically enhancing phytoremediation of heavy metal-contaminated soil using nitrogen-sulfur-doped carbon quantum dots and endophytic bacteria. The method involves planting remediation plants in heavy metal-contaminated soil, applying nitrogen-sulfur-doped carbon quantum dots and endophytic bacteria for cultivation, and then completing the remediation process. This invention, by constructing a material-microorganism-plant synergistic remediation system, not only improves the absorption and translocation efficiency of heavy metals by remediation plants, overcoming the bottleneck of phytoremediation efficiency, but also enhances the resilience of remediation plants under heavy metal stress conditions. Simultaneously, it improves the rhizosphere microenvironment, promotes the enrichment of beneficial microorganisms, and thus helps maintain the long-term remediation effect of the soil. This method not only improves the efficiency of phytoremediation of heavy metal-contaminated soil but also enhances the adaptability of plants under heavy metal stress conditions, improves the rhizosphere microenvironment, and maintains soil microecological stability, demonstrating good application prospects and promotional value.

Description

Technical Field

[0001] This invention belongs to the field of soil remediation technology and relates to a method for synergistically enhancing phytoremediation of heavy metal contaminated soil by using nitrogen-sulfur doped carbon quantum dots and plant endophytic bacteria. Background Technology

[0002] Among the remediation technologies for heavy metal contaminated soil, phytoremediation is considered a relatively economical and environmentally friendly method due to its advantages of eliminating secondary pollution, low cost, and suitability for large-scale application. However, existing phytoremediation technologies still suffer from problems in practical applications, such as unstable remediation efficiency, long remediation cycles, and susceptibility to environmental conditions. Furthermore, the variety of heavy metal hyperaccumulating plants is limited, while non-hyperaccumulating plants generally suffer from low heavy metal absorption efficiency, small biomass, and poor tolerance. These factors, to varying degrees, restrict the promotion and application of phytoremediation technologies.

[0003] Existing research has shown that certain functional nanomaterials can influence plant growth, physiological metabolism, and heavy metal absorption processes under specific conditions, thus helping to alleviate heavy metal stress to some extent. However, relying solely on a single material or a single phytoremediation method often fails to simultaneously address the multiple needs of plant growth, efficient heavy metal absorption, and soil ecological stability. In particular, the following shortcomings are still prevalent in practical applications: (a) high requirements for plant tolerance, limiting adaptability and effectiveness; (b) limited promotion of plant growth, making it difficult to significantly increase plant biomass; (c) insufficient capacity for heavy metal absorption and accumulation, requiring further optimization of remediation efficiency and cycle; and (d) long-term application of a single remediation method may adversely affect soil microbial structure.

[0004] Therefore, there is an urgent need to develop a new synergistic remediation method that can ensure the efficiency of phytoremediation while taking into account plant growth, stress resistance, and soil microecological stability. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the shortcomings of the prior art and provide a method for enhancing the phytoremediation of heavy metal contaminated soil by using nitrogen and sulfur doped carbon quantum dots and plant endophytic bacteria. By constructing a material-microorganism-plant synergistic remediation system, it can not only improve the absorption and translocation efficiency of heavy metals by remediation plants and break through the bottleneck of phytoremediation efficiency, but also enhance the stress resistance of remediation plants under heavy metal stress conditions (such as antioxidant capacity), while improving the rhizosphere microenvironment of plants and promoting the enrichment of beneficial microorganisms, thereby helping to maintain the long-term remediation effect of the soil.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A method for synergistically enhancing phytoremediation of heavy metal contaminated soil using nitrogen-sulfur doped carbon quantum dots and endophytic bacteria, the method comprising the following steps: Planting remediation plants in heavy metal-contaminated soil, applying nitrogen-sulfur-doped carbon quantum dots and endophytic bacteria to cultivate the remediation plants, and completing the remediation of heavy metal-contaminated soil.

[0007] In a further improvement to the above method, the plant endophytic bacteria are applied to the roots of the repaired plant via root irrigation.

[0008] In a further improvement to the above method, the amount of plant endophytic bacteria added is 2 mL of the plant endophytic bacteria applied to the roots of each of the repaired plants.

[0009] In a further improvement to the above method, the plant endophytic bacteria are applied in the form of a bacterial solution with an OD600 of 1.0.

[0010] In a further improvement to the above method, the plant endophytic bacteria are applied once every two weeks.

[0011] In a further improvement to the above method, the plant endophytic bacteria are endophytic bacteria with heavy metal tolerance and / or plant growth-promoting functions.

[0012] A further improvement to the above method is that the plant endophytic fungus is... Enterobacter sp. YG-14, but not limited to that.

[0013] In a further improvement to the above method, the remediation plant is a plant that has the ability to accumulate or tolerate heavy metals.

[0014] A further improvement to the above method is that the remediation plant is *Sedum aizoon* (also known as mineral-bearing sedum). Sedum plumbizincicola However, this is not the only one.

[0015] The above method is further improved by the following method: the application of nitrogen-sulfur-doped carbon quantum dots includes at least one of the following: soil application, foliar spraying, or a combination of both.

[0016] A further improvement to the above method is that, when applied to soil, the amount of nitrogen-sulfur-doped carbon quantum dots is 100 mg to 5000 mg per kilogram of the heavy metal-contaminated soil. Specifically, the application amounts of nitrogen-sulfur-doped carbon quantum dots can be 100 mg / kg, 300 mg / kg, 500 mg / kg, 800 mg / kg, 1000 mg / kg, 1200 mg / kg, 1500 mg / kg, 1800 mg / kg, 2000 mg / kg, 2100 mg / kg, 2200 mg / kg, 2500 mg / kg, 2800 mg / kg, 3000 mg / kg, 3100 mg / kg, 3200 mg / kg, 3500 mg / kg, 3800 mg / kg, 4000 mg / kg, 4200 mg / kg, 4500 mg / kg, 4800 mg / kg, and 5000 mg / kg, but are not limited to these.

[0017] A further improvement to the above method is that, when using foliar spraying, the application concentration of the nitrogen-sulfur-doped carbon quantum dots is 10 mg / L to 500 mg / L. For example, the application concentration of the nitrogen-sulfur-doped carbon quantum dots can be 10 mg / L, 20 mg / L, 40 mg / L, 50 mg / L, 70 mg / L, 80 mg / L, 90 mg / L, 100 mg / L, 110 mg / L, 120 mg / L, 130 mg / L, 140 mg / L, 150 mg / L, 170 mg / L, 180 mg / L, 190 mg / L, 200 mg / L, 210 mg / L, 220 mg / L, 240 mg / L, 250 mg / L, 270 mg / L, 280 mg / L, 290 mg / L, 300 mg / L, 310 mg / L, 320 mg / L, 340 mg / L, 350 mg / L, etc. The concentrations of nitrogen-sulfur-doped carbon quantum dots are 370 mg / L, 380 mg / L, 390 mg / L, 400 mg / L, 410 mg / L, 420 mg / L, 440 mg / L, 450 mg / L, 470 mg / L, 480 mg / L, 490 mg / L, and 500 mg / L, but not limited to these. The single application volume of the nitrogen-sulfur-doped carbon quantum dots is 1.5 mL to 5 mL, for example, 1.5 mL, 2 mL, 2.5 mL, 3 mL, 3.5 mL, 4 mL, 4.5 mL, and 5 mL, but not limited to these. The foliar spraying cycle of the nitrogen-sulfur-doped carbon quantum dots is 1 to 3 times per week, for example, 1 to 2 times per week, and 3 times per week, but not limited to these.

[0018] The above method is further improved in that the nitrogen-sulfur-doped carbon quantum dots are prepared by hydrothermal reaction using carbon-containing precursors and nitrogen- and sulfur-containing precursors as raw materials. Specifically, the carbon-containing precursors, nitrogen- and sulfur-containing precursors and water are mixed and subjected to hydrothermal reaction to obtain crude nitrogen-sulfur-doped carbon quantum dots.

[0019] In a further improvement to the above method, the mass ratio of the carbon-containing precursor to the nitrogen- and sulfur-containing precursor is 0.64:1.08.

[0020] In a further improvement to the above method, the carbon-containing precursor is citric acid, but it is not limited to this; for example, it can also be malic acid, tartaric acid, or glucose.

[0021] In a further improvement to the above method, the nitrogen- and sulfur-containing precursor is L-cysteine, but it is not limited to this; for example, it can also be methionine or thioacetamide.

[0022] In a further improvement to the above method, the hydrothermal reaction temperature is 160 ℃~220 ℃, and the reaction time is 4h~10h.

[0023] The above method is further improved by including the following treatment after the hydrothermal reaction: sonicating, filtering, purifying, freezing, and drying the product obtained after the hydrothermal reaction (crude nitrogen-sulfur-doped carbon quantum dots); the sonication time is 30 min to 60 min; the filtration uses a filter membrane with a pore size of 0.22 μm; the purification time is 48 h to 72 h, and the purification is performed using a dialysis bag with a molecular weight cutoff of 500 Da; the freezing temperature is -150℃ to -80℃, and the freezing time is 12 h to 48 h; the drying is vacuum freeze-drying.

[0024] The above method is further improved so that the initial concentration of heavy metals in the heavy metal-contaminated soil is ≤100mg / kg.

[0025] The above method is further improved in that the heavy metals in the heavy metal-contaminated soil include at least one of cadmium, lead, zinc, and copper.

[0026] In a further improvement to the above method, the culture time is ≥2 weeks.

[0027] Compared with the prior art, the advantages of the present invention are as follows: This invention proposes a method for enhancing phytoremediation of heavy metal contaminated soil by synergistically combining nitrogen-sulfur-doped carbon quantum dots (such as N,S-CDs) and plant endophytic bacteria (such as YG-14). The synergistic effect of these two methods yields the following unexpected technical benefits: (1) Compared with the application of N,S-CDs or YG-14 alone, the synergistic treatment of N,S-CDs and YG-14 in this invention can significantly improve the efficiency of phytoremediation of heavy metals in soil.

[0028] (2) Compared with the application of N,S-CDs or YG-14 alone, the synergistic treatment of N,S-CDs and YG-14 in this invention can significantly improve the plant growth rate, enhance stress resistance, and improve the plant’s absorption of heavy metal cadmium in the soil.

[0029] (3) Compared with the application of N,S-CDs or YG-14 alone, the synergistic treatment of N,S-CDs and YG-14 in this invention is beneficial to the restoration of soil ecological function and the increase of the relative abundance of beneficial bacteria in heavy metal soil. (4) Compared with the application of N,S-CDs or YG-14 alone, the present invention significantly enhances the applicability and stability of the remediation system in hyperaccumulating plants by synergistic treatment of N,S-CDs and YG-14, proving that the synergistic strategy can still effectively promote plant growth and improve the absorption and translocation efficiency of heavy metals under hyperaccumulating plant conditions, and its comprehensive remediation effect is significantly better than the application of N,S-CDs or YG-14 alone.

[0030] Therefore, the synergistic remediation method provided by this invention can not only improve the efficiency of phytoremediation of heavy metal contaminated soil, but also enhance the adaptability of plants under heavy metal stress conditions, while improving the plant rhizosphere microenvironment and maintaining soil microecological stability, and has good application prospects and promotion value. Attached Figure Description

[0031] Figure 1 This is a comparison diagram showing the effects of different treatments on the growth of *Sedum aizoon* under cadmium stress in Example 1 of this invention.

[0032] Figure 2 This is a comparison chart showing the nutrient element absorption of *Sedum aizoon* under cadmium stress under different treatment conditions in Example 1 of the present invention.

[0033] Figure 3 This is a comparison chart showing the cadmium absorption and accumulation in Sedum aizoon under different treatment conditions in Example 1 of the present invention.

[0034] Figure 4 This is a comparison diagram of the photosynthetic status of Sedum sarmentosum under different treatment conditions in Example 1 of the present invention.

[0035] Figure 5 This is a comparison chart of the antioxidant properties of Sedum sarmentosum under different treatment conditions in Example 1 of the present invention.

[0036] Figure 6This is a comparison diagram of the physicochemical properties of the rhizosphere soil of Sedum sarmentosum under different treatment conditions in Example 1 of the present invention. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but this does not limit the scope of protection of the present invention. All materials and instruments used in the following embodiments are commercially available.

[0038] Example 1: A method for synergistically enhancing phytoremediation of heavy metal contaminated soil using nitrogen-sulfur-doped carbon quantum dots (N,S-CDs) and plant endophytic bacteria (YG-14) is disclosed. This method involves planting a remediation plant (the hyperaccumulating plant *Sedumplum bizincicola*) in cadmium-contaminated soil, applying N,S-CDs, and using YG-14 as endophytic bacteria, and then cultivating the plant. The synergistic effect of N,S-CDs and YG-14 promotes the remediation of heavy metal contaminated soil by *Sedumplum bizincicola*. The N,S-CDs are prepared from citric acid and L-cysteine ​​through a hydrothermal reaction and purification process, as detailed below. (1) Experimental materials Experimental plant: The remediation plant used was Sedum plumbizincicola.

[0039] Experimental strains: the plant endophytes used Enterobacter sp. YG-14 (hereinafter referred to as YG-14) is currently deposited at the China General Microbiological Culture Collection Center (CGMCC) with the preservation number CGMCC No. 15107. Those skilled in the art can obtain this strain based on this preservation number.

[0040] Specifically, the cultivation method for YG-14 includes the following steps: Remove the YG-14 bacterial culture from the -80 ℃ ultra-low temperature freezer. Use an inoculation loop to take a portion of the bacterial suspension and streak it onto LB solid medium. Incubate in an inverted position at 30 ℃ for 12-24 h to obtain plates containing single YG-14 colonies. Use an inoculation loop to pick up a single YG-14 colony and place it on LB liquid medium. Incubate on a constant temperature shaker at 170 rpm and 30 ℃ to obtain the YG-14 bacterial suspension. Measure the absorbance of the YG-14 bacterial suspension at 600 nm using a UV spectrophotometer. An absorbance of approximately 1.0 indicates the optimal inoculation concentration for plant inoculation.

[0041] Experimental soil: Original soil from uncontaminated farmland in Qingjiangpu District, Huai'an City, Jiangsu Province (119°01′53″E, 33°61′04″N), with a cadmium content of 0.108 mg / kg. The screened soil was divided into 2.5 kg portions, and 10 mg / kg of cadmium was artificially added. Water was added to the soil to 70% of its maximum water-holding capacity, and it was mixed with an equal volume of washed quartz sand (v:v=1:1). The mixture was then left to stand at a constant temperature (22-24℃) for 2 months to reach equilibrium and promote the adsorption of the added cadmium.

[0042] In this embodiment, the preparation method of nitrogen-sulfur-doped carbon quantum dots (N,S-CDs) includes the following steps: 1) 0.64 g citric acid, 1.08 g L-cysteine ​​and 60 mL deionized water were placed in a 100 mL polytetrafluoroethylene-lined high-temperature reactor and reacted in an oven at 180 °C for 6 h. After cooling to room temperature, the crude product of nitrogen-sulfur-doped carbon quantum dots was obtained.

[0043] 2) The crude nitrogen-sulfur-doped carbon quantum dots were sonicated for 30 min to disperse them uniformly in an aqueous solution. Then, the solution was filtered through a 0.22 μm filter membrane and purified in a dialysis bag with a molecular weight cutoff of 500 Da for 48 hours. The purified solution was then frozen overnight (24 h) at -80 °C. Finally, the solution was freeze-dried under vacuum to obtain nitrogen-sulfur-doped carbon quantum dots, denoted as N,S-CDs.

[0044] (2) Experimental methods In this experiment, cadmium-contaminated soil was used. The soil was evenly distributed into each experimental flowerpot, with each pot containing approximately 250 g of soil. The *Sedum aizoon* cuttings were washed with tap water, cut into uniformly sized branches, leaving only two leaves at the top and removing the rest. These cuttings were then transplanted into the experimental flowerpots, with three seedlings planted in each pot. After the seedlings had grown for three weeks and developed new leaves, the *Sedum aizoon* in each group were treated according to the following methods. (3) Experimental grouping 1) Cadmium-free soil control group (CCK): The experiment was conducted using uncontaminated cadmium soil (cadmium concentration of 0 mg / kg).

[0045] 2) Control group (CK): Cadmium-contaminated soil (cadmium concentration of 10 mg / kg) was used in the experiment.

[0046] 3) N,S-CDs application group (CDs): Cadmium-contaminated soil (cadmium concentration of 10 mg / kg) was used for the experiment. 300 mg / kg N,S-CDs aqueous solution was added to each pot and mixed into the soil. The other groups were given an equal amount of deionized water.

[0047] 4) YG-14 application group (YG-14): The experiment was conducted using cadmium-contaminated soil (cadmium concentration of 10 mg / kg). 2 mL of YG-14 bacterial solution (OD600=1.0) was applied to the roots of each Sedum sarmentosum plant, once every two weeks, for a total of four applications and eight weeks. The other groups were treated with the same amount of deionized water.

[0048] 5) YG-14 and N,S-CDs combined application group (Y+CDs): Cadmium-contaminated soil (cadmium concentration of 10 mg / kg) was used for the experiment. 300 mg / kg N,S-CDs aqueous solution was added to each pot and mixed into the soil. The other groups were treated with an equal amount of deionized water. 2 mL of YG-14 bacterial solution (OD600=1.0) was applied to the roots of each Sedum sarmentosum plant every two weeks for four weeks. The other groups were treated with an equal amount of deionized water.

[0049] Each group had 5 replicates. This experiment was conducted in a greenhouse with a photoperiod of 16 / 8 h (light / dark) and a light intensity of 500 μmol / m². -2 s -2 Water the plants weekly as needed to maintain their normal growth.

[0050] Figure 1 This is a comparison diagram showing the effects of different treatments on the growth of *Sedum aizoon* under cadmium stress in Example 1 of this invention. Figure 1 In the following categories, (a) plant height, (b) root length, (c) fresh weight of leaves, (d) fresh weight of stems, (e) fresh weight of roots, (f) dry weight of leaves, (g) dry weight of stems, and (h) dry weight of roots.

[0051] like Figure 1As shown, compared to the CK group, the plant height of the CCK group, CDs group, YG-14 group, and Y+CDS group increased by 63.24%, 36.03%, 44.26%, and 58.09%, respectively; the root length increased by 128.66%, 32.93%, 54.88%, and 81.40%, respectively; the fresh weight of leaves increased by 282.15%, 142.12%, 131.82%, and 191.27%, respectively; the fresh weight of stems increased by 149.34%, 70.19%, 69.74%, and 107.05%, respectively; and the fresh weight of roots increased by [missing data]. The plant height, root length, and other indicators of the CCK, CDs, YG-14, and Y+CDS groups increased by 247.81%, 166.67%, 178.45%, and 215.26%, respectively; leaf dry weight increased by 231.73%, 122.22%, 127.13%, and 176.48%, respectively; stem dry weight increased by 116.45%, 55.48%, 66.45%, and 86.77%, respectively; and root dry weight increased by 294.07%, 207.63%, 198.31%, and 263.56%, respectively. This shows that compared to the CK group, the plant height, root length, and other indicators of the CCK, CDs, YG-14, and Y+CDS groups all increased significantly, indicating that soil cadmium stress significantly inhibits the growth of *Sedum aizoon*. However, the single and combined application of N,S-CDs and YG-14 can alleviate the growth inhibition effect of cadmium stress on *Sedum aizoon*, and the combined application is more effective.

[0052] Figure 2 This is a comparison chart showing the nutrient element absorption of *Sedum aizoon* under cadmium stress under different treatment conditions in Example 1 of the present invention. Figure 2 In the following, (a) iron content, (b) sulfur content, and (c) phosphorus content.

[0053] Depend on Figure 2It can be seen that, compared with the CK group, the iron content of each Sedum aizoon plant in the CCK group, CDs group, YG-14 group and Y+CDS group increased by 278.62%, 139.68%, 109.69% and 226.17%, respectively; the sulfur content increased by 164.37%, 121.60%, 164.81% and 192.18%, respectively; and the phosphorus content increased by 232.01%, 116.71%, 113.32% and 127.00%, respectively. This shows that the iron, sulfur, and phosphorus contents of *Sedum aizoon* in the CCK, CDs, YG-14, and Y+CDS groups were significantly higher than those in the CK group, indicating that soil cadmium stress inhibits the absorption of nutrients by *Sedum aizoon*. However, the single and combined application of N,S-CDs and YG-14 can promote the absorption of nutrients such as iron, sulfur, and phosphorus by *Sedum aizoon*, with the combined application showing better results. This corresponds to the growth index results, indicating that the combined application of N,S-CDs and YG-14 further promotes the absorption of nutrients by plants, thereby promoting plant growth.

[0054] Figure 3 This is a comparison chart showing the cadmium absorption and accumulation in Sedum aizoon under different treatment conditions in Example 1 of the present invention. Figure 3 In the figure, (a) cadmium content in leaves, (b) cadmium content in stems, and (c) cadmium content in roots.

[0055] Depend on Figure 3 It can be seen that the cadmium content in the CCK group was approximately 0 μg / plant. -1 The cadmium content in the leaves of the *Sedum aizoon* group was around 134.70%, while the cadmium content in the other groups was relatively high, indicating that *Sedum aizoon* absorbs and accumulates cadmium in cadmium-containing soil. Furthermore, compared to the control group, the cadmium content in the leaves of the CDs, YG-14, and Y+CDS groups increased by 134.70%, 146.68%, and 206.99%, respectively; the cadmium content in the stems increased by 54.96%, 68.08%, and 96.71%, respectively; and the cadmium content in the roots increased by 148.80%, 127.22%, and 184.90%, respectively. These results indicate that both single and combined application of N,S-CDs and YG-14 can promote the absorption of cadmium in the roots, stems, and leaves of *Sedum aizoon*, with the combined application showing a better effect. This suggests that N,S-CDs and YG-14 synergistically promote the absorption and accumulation of cadmium in *Sedum aizoon*, further improving the efficiency of cadmium pollution remediation in the soil.

[0056] Figure 4 This is a comparison diagram of the photosynthetic status of Sedum sarmentosum under different treatment conditions in Example 1 of the present invention. Figure 4 In the figure, (a) total chlorophyll content, (b) nitrogen content, (c) chlorophyll a content, (d) chlorophyll b content, and (e) xanthophyll content.

[0057] Depend on Figure 4 It was found that, compared with the CK group, the total chlorophyll content of the CCK group, CDs group, YG-14 group, and Y+CDS group increased by 16.96%, 13.44%, 26.17%, and 16.90%, respectively; nitrogen content increased by 13.45%, 10.32%, 20.80%, and 13.14%, respectively; chlorophyll a content increased by 25.92%, 35.34%, 64.92%, and 62.57%, respectively; chlorophyll b content increased by 17.43%, 18.35%, 53.52%, and 58.41%, respectively; and xanthophyll content increased by 119.35%, 138.71%, 67.74%, and 87.10%, respectively. These results indicate that cadmium stress affects plant photosynthesis, while the single and combined application of N,S-CDs and YG- All 14 can alleviate the inhibition of plant photosynthesis caused by cadmium stress.

[0058] Figure 5 This is a comparison chart of the antioxidant properties of Sedum sarmentosum under different treatment conditions in Example 1 of the present invention. Figure 5 In the figure, (a) SOD enzyme activity, (b) POD enzyme activity, (c) CAT enzyme activity, (d) MDA content, and (e) H2O2 content.

[0059] Figure 5 Several enzyme activity indicators related to antioxidant properties were measured. Figure 5It can be seen that, compared with the CK group, the SOD enzyme activity of the CCK group, CDs group, YG-14 group and Y+CDS group increased by 572.20%, 154.74%, 247.28% and 266.54%, respectively; the POD enzyme activity increased by 365.66%, 708.19%, 572.53% and 881.81%, respectively; the CAT enzyme activity increased by 2.15%, 2.88%, 1.64% and 5.55%, respectively; the MDA content decreased by 38.83%, 30.93%, 20.29% and 22.32%, respectively; and the H2O2 content decreased by 17.33%, 22.50%, 31.35% and 33.03%, respectively. Compared to the CCK group, the CK, CDs, YG-14, and Y+CDS groups showed significantly decreased SOD, POD, and CAT enzyme activities, and significantly increased H2O2 and MDA contents. These results indicate that cadmium stress in soil reduces the activity of antioxidant enzymes in plants, hindering their ability to effectively remove excess ROS and producing more H2O2 and MDA, thus increasing oxidative stress and causing oxidative damage. In contrast, the application of N,S-CDs and YG-14 helps plants cope with oxidative stress and damage caused by cadmium stress by enhancing antioxidant enzyme activity and reducing oxidative stress indicators; the combined application yields even better results.

[0060] Figure 6 This is a comparison diagram of the physicochemical properties of the rhizosphere soil of Sedum sarmentosum under different treatment conditions in Example 1 of the present invention. Figure 6 Among them, (a) total cadmium content in soil, (b) cadmium content in soil with weak acid extractability, (c) soil pH, (d) soil CEC content, and (e) soil organic matter content.

[0061] Figure 6 The effects of N, S-CDs and YG-14 on the rhizosphere soil of Sedum sarmentosum with mineral content were investigated, and the physicochemical properties of the rhizosphere soil of each group of Sedum sarmentosum with mineral content were measured in the eighth week.

[0062] Depend on Figure 6 (a) Figure 6(b) It can be seen that, compared with the CK group, the total cadmium content in the soil of the CDs group, YG-14 group, and Y+CDS group decreased by 6.21%, 5.80%, and 9.47%, respectively, and the content of weakly acid-extractable cadmium decreased by 9.30%, 11.68%, and 25.46%, respectively. This indicates that the application of N,S-CDs and YG-14 alone and in combination reduced the total cadmium content in the soil, enhanced the remediation effect of Sedum aizoon on cadmium pollution in the soil, and the combined application was more effective. At the same time, the content of weakly acid-extractable cadmium in the soil also decreased. Weakly acid-extractable cadmium is a form of cadmium that can be absorbed and utilized by plants, indicating that the application of N,S-CDs and YG-14 alone and in combination promoted the absorption of weakly acid-extractable cadmium by Sedum aizoon, and the combined application was more effective.

[0063] Depend on Figure 6 (c) It can be seen that the CDs group has the lowest pH value, which may be because the N,S-CDs surface has functional groups such as -C-OH and -OH, which will dissociate into H+. + This leads to a decrease in pH value; Figure 6 (d) shows the changes in soil CEC content. (From...) Figure 6 (d) It can be seen that, compared with the CK group, the soil CEC content of the CDs group, YG-14 group, and Y+CDS group increased by 1.43%, 2.81%, 3.85%, and 6.23%, respectively. Increased soil CEC content usually indicates improved soil quality. It was found that both N,S-CDs and YG-14 significantly increased soil CEC content and improved soil quality, and the combined application of N,S-CDs and YG-14 had a more pronounced effect.

[0064] Depend on Figure 6 (e) It can be seen that the soil organic matter content of each group remained basically unchanged.

[0065] Investigating the synergistic effects of N,S-CDs and YG-14 on the rhizosphere soil microbial community of Sedum aizoon under cadmium stress. When the Sedum sarmentosum in the third treatment reached the eighth week of growth, the plants were removed from the pots with soil attached. Large clumps of soil were shaken off, and residual soil was collected from the roots using a sterile brush. 3-5 g of rhizosphere soil from each treatment group was placed in cryovials. At least 3 samples were collected from each treatment, flash-frozen in liquid nitrogen for more than 1 hour, and stored in a -80 ℃ freezer.

[0066] The study examined different treatment conditions (CCK group, CK group, CDs group, YG-14 group, and Y+CD). SBacterial dilution curves and Shannon index curves were obtained from the rhizosphere soil of *Sedum morganianum* (group 1). Bacterial dilution curves and Shannon index curves are statistical tools used to assess whether sequencing data accurately reflects species diversity. The results showed that both the bacterial dilution curves and the Shannon index curves eventually flattened out, indicating that the collected samples were accurate and reliable, and that the sequencing data essentially covered all bacterial communities in the soil samples from each group, demonstrating high reliability.

[0067] The study examined different treatment conditions (CCK group, CK group, CDs group, YG-14 group, and Y+CD). S The Venn diagram of rhizosphere soil bacteria in *Sedum aizoon* (group 1) can visually present the relationship of ASVs among the five groups. The results showed that the CCK group had 2587 ASVs, the CK group had 1873 ASVs, the CDs group had 1702 ASVs, the YG-14 group had 766 ASVs, and the Y+CDs group had 874 ASVs. The CCK, CK, CDs, YG-14, and Y+CDs groups also showed a significant difference in ASV counts. S The groups shared a total of 653 ASVs. These results indicate that the CCK group in cadmium-free soil had the highest number of ASVs, while the number of ASVs specific to the cadmium-containing soil groups was lower than that in the CCK group, suggesting that cadmium pollution affects the species richness of soil bacterial communities. Furthermore, the bacterial communities among the different treatment groups shared some ASVs, indicating that some bacterial groups in the soil have broad adaptability and can survive in complex and variable soil environments.

[0068] The study examined different treatment conditions (CCK group, CK group, CDs group, YG-14 group, and Y+CD). S Principal Coordinate Analysis (PCoA) and Non-Metric Multidimensional Scale (NMDS) plots of bacteria in the rhizosphere soil of *Sedum morganianum* (group 1) were used to illustrate the relationships between soil microbial communities in different treatment groups. The results showed that the CCK group was clearly separated from other cadmium-contaminated treatment groups, indicating that cadmium pollution significantly altered the soil bacterial community structure. The CDs, YG-14, and Y+CDs groups were spaced apart from each other and differed from the CK group, indicating that the individual and combined application of N, SCDs, and YG-14 had different effects on the soil bacterial community structure. Specifically, the PCoA plots showed that the YG-14 and Y+CDs groups were relatively close, with some overlap, indicating that the bacterial community structures of the YG-14 and Y+CDs groups were quite similar.

[0069] LEfSe analysis can provide in-depth insights into the differences in soil microbial communities across different groups, identifying microbial groups that exhibit significant differences under varying soil conditions. These differentially expressed groups may be closely related to treatment methods, soil environmental factors, and other factors, providing important clues for revealing the interaction mechanisms between soil microorganisms and the soil environment. LEfSe analysis (LDA value > 3) was performed on the rhizosphere soil bacterial communities of *Sedum aizoon* in different treatment groups, and the results are as follows: The top 50 bacterial groups with the most significant inter-group differences among the various treatment groups of Sedum sarmentosum.

[0070] The LEfSe analysis results of the differential bacterial groups show that the labeled group name indicates that its relative abundance in the corresponding group is significantly higher than that in other groups.

[0071] The results from the evolutionary branching diagram show that: The CCK group identified 82 differentially classified bacterial taxa, with 19 of them ranking in the top 50. Excluding unclassified taxa, these are Cyanobacteria, Cyanobacteriia, Chloroflexi, Actinobacteriota, Micrococcales, and Micrococcaceae. Arthrobacter , Arthrobacter _sp._NW-2013-Rh11, Chloroflexia, Burkholderiales, Leptolyngbyales, Leptolyngbyaceae, Cyanobacteriales, Coleofasciculaceae, Microcoleus_Es-Yyy1400 Anaerolineae and Burkholderiales were relatively abundant in cadmium-free soils, indicating that the relative abundance of Anaerolineae and Burkholderiales was significantly reduced, and the relative abundance of Chloroflexi was significantly reduced in cadmium-containing soils.

[0072] A total of 148 differentially expressed bacterial taxa were identified in the CK group, with 13 ranking in the top 50: Alphaproteobacteria, Rhizobiales, Rhizobiaceae, Pseudomonadales, Gemmatimonadota, Acidobacter eriota, Firmicutes, Bacteroidota, and Bacteroidia. Ensifer , Ensifer_ adhaerensThe relative abundance of groups such as Gemmatimonadetes and Gemmatimonadales was significantly increased in the CK group, suggesting that they may have strong cadmium resistance. This indicates that the relative abundance of Acidobacteriota was significantly increased in cadmium-contaminated soil. The relative abundance of Gemmatimonas under Bacteroidota and Gemmatimonadota increased with the increase of cadmium concentration in the soil. Firmicutes and Bacteroidota were the dominant bacterial phyla before cadmium-contaminated soil remediation, all indicating that these groups have cadmium resistance.

[0073] The CDs group identified 89 differentially classified bacterial groups, with 9 ranking in the top 50. Excluding unclassified groups, these are Actinobacteriota, Actinobacteria, Micrococcales, Thermomicrobiales, and Cellulomonadaceae. Actinotalea , Pirellula These taxa may possess cadmium resistance, and N,S-CDs contribute to their relative abundance, indicating that Actinobacteria can produce phosphate-solubilizing agents, fix nitrogen, produce plant growth hormones to promote plant growth, and improve soil ecology through multiple mechanisms, including producing chitinase to decompose fungal cell walls and inhibit pathogen growth. This suggests that N,S-CDs enrich some bacterial taxa that improve soil ecology and promote plant growth. Thermomicrobiales are a class of microorganisms found in extreme environments, typically high temperatures, high salinity, or other extreme conditions, suggesting that Thermomicrobiales may have the ability to tolerate extreme environments, a characteristic that may enable them to function under heavy metal cadmium stress.

[0074] Four differentially classified bacterial groups were identified in the YG-14 group. After removing unclassified bacterial groups, the results were as follows: Planomicrobium ,Planococcaceae Planomicrobium_chinense These groups may be cadmium resistant, and YG-14 helps to increase their relative abundance, indicating that Planococcaceae has cadmium tolerance and can promote plant growth. It shows tolerance to a variety of heavy metals such as arsenic, zinc, cadmium, chromium, copper, cobalt, mercury and lead, and can survive in high concentrations of these heavy metals. At the same time, Planococcaceae bacteria can grow in nitrogen-free medium, indicating that they have nitrogen-fixing ability, which may provide nitrogen nutrition for plants and promote plant growth.

[0075] The Y+CDs group identified nine differentially expressed bacterial taxa, all of which ranked in the top 50: Proteobacteria, Gammaproteobacteria, Enterobacteriaceae, Enterobacterales, and others. Klebsiella , Klebsiella_aerogenes Erwiniaceae Pantoea , Pantoea_agglomerans These groups may be cadmium resistant, and the combined application of YG-14 and N,S-CDs helps to increase their relative abundance. This indicates that the relative abundance of Proteobacteria increases significantly in cadmium-contaminated soil, and Proteobacteria is the group of bacteria with the most abundant heavy metal resistance genes, which can regulate multiple pathways to reduce cadmium toxicity.

[0076] The results of the differential bacterial genera relative abundance in the rhizosphere microbial community of *Sedum morganianum* show that: CCK Group Arthrobacter It has the effect of promoting plant growth and improving plant resistance to heavy metals, for example, Arthrobacter sp. PGP41 is a cadmium-resistant bacterium isolated from the rhizosphere soil of cadmium hyperaccumulating plants, which can improve the growth of plant seedlings and roots under cadmium stress. Arthrobacter sp. GN70 can promote rice growth; Arthrobacter nicotinovorans JI39 can significantly promote ginseng growth, upregulate the expression of genes such as SOD in ginseng, and increase the activity of urease and invertase in the soil. However, high concentrations of heavy metals can inhibit its growth. Arthrobacter The abundance was relatively high in the CCK and CDs groups, indicating that cadmium stress inhibited its growth, but the absence of cadmium or the application of N,S-CDs promoted its growth. Arthrobacter An increase in relative abundance. Microcoleus_Es-Yyy1400 Related to plant resistance Microcoleus The abundance was higher in undegraded ecosystems and lower in highly degraded ecosystems, indicating that... [[ID=5 Abundance decreases in harsh environments, but increases as environmental conditions improve.

[0077] CK Group ​ It is a cadmium-resistant bacterium that promotes plant growth and removes cadmium. ​ ​ The CK group can tolerate high concentrations of cadmium and still secrete substances that promote plant growth, such as IAA and siderophores, even in culture media containing heavy metals. This indicates that the CK group accumulates some cadmium-resistant bacteria under cadmium stress, which helps Sedum sarmentosum resist cadmium stress in cadmium-polluted environments.

[0078] CDs group​ It can grow in harsh conditions, and fertilization can further promote its growth. For example, ​ The isolation of sp. nov. from iron ore powder suggests that bacteria in this genus may be able to survive in heavy metal environments; ​ It is the dominant bacterial genus in oil palm plantation soils. Its relative abundance is relatively high in soils that have been treated with inorganic fertilizers for a long time. Long-term application of inorganic fertilizers may increase its relative abundance by changing the chemical properties of the soil. ​ These bacteria can utilize carbon sources in petroleum for growth under both aerobic and anaerobic conditions, thereby participating in the carbon cycle during petroleum degradation. ​ Fertilization can also promote an increase in their relative abundance. These results indicate that N,S-CDs application provides additional nitrogen, carbon, and other nutrients to certain bacterial groups, thereby promoting an increase in the relative abundance of these groups and potentially helping to improve soil physicochemical properties.

[0079] YG-14 group ​ It can also tolerate cadmium pollution and promote plant growth, for example... ​ Strain P1 can survive in heavy metal environments and tolerate a variety of heavy metals. It can promote sunflower growth under drought stress, increase the content of chlorophyll, sugar and phenols in leaves, and reduce the content of leaf proline and malondialdehyde. When treated with salicylic acid, it can effectively reduce the inhibition of plant growth and development by heavy metals such as cadmium and enhance the accumulation of cadmium in sunflowers. ​ The strain S5-TS A-19 exhibits tolerance to the toxic environment of HMX, suggesting it may also be cadmium tolerant. This indicates that application of YG-14 enriches beneficial bacteria that are tolerant to cadmium pollution and promote plant growth, thus helping *Sedum aizoon* to survive better in cadmium-contaminated environments.

[0080] Y+CDs group ​ It has the ability to tolerate cadmium pollution, improve soil ecology, and promote plant growth, for example... ​ Cadmium resistance gene ​ and ​ It exhibits strong cadmium resistance and can also produce bio-flocculation agents to adsorb cadmium, thereby removing cadmium from aqueous solutions; simultaneously ​ The strain can also produce IAA, siderophores, etc., and has the ability to dissolve phosphate. It can significantly increase plant height, root length and chlorophyll content, and reduce H2O2 levels. At the same time, it can promote the adsorption, chelation or precipitation of cadmium in the soil, thereby reducing cadmium toxicity. ​ JLS50 can increase the concentration of acid-soluble cadmium in soil and reduce the concentration of reduced, oxidized and residual cadmium. ​ R3-3 can produce IAA and dissolve phosphates and has cadmium tolerance, which can reduce the cadmium content in rice plants;​ ​ G129K1-1 can significantly promote rapeseed growth and enhance the plant's absorption and translocation of cadmium. ​ It also possesses the ability to tolerate cadmium pollution, improve soil ecology, and promote plant growth, for example... ​ ​ MCC3089 has a high cadmium removal efficiency, up to 97%. Under cadmium stress, it can improve the germination rate of rice seeds, promote the growth of rice seedlings, and increase chlorophyll content, antioxidant enzyme activity, proline content, and reduce malondialdehyde content. ​ (HR1) can absorb cadmium through ion exchange and physical adsorption; ​ It can convert cadmium into inert CdS through biotransformation, thus exhibiting tolerance and resistance to cadmium toxicity. ​ It can still grow in cadmium-containing culture medium, and can dissolve calcium phosphate to release phosphate ions, which react with lead and cadmium in the soil to form more stable compounds, thereby reducing the bioavailability of heavy metals. ​ ​ M2 is a phosphate-solubilizing bacterium that secretes organic acids to dissolve insoluble phosphates in the soil, converting them into phosphorus available to plants. It can also induce cadmium phosphate precipitation, reducing cadmium concentration. Simultaneously, it promotes wheat growth under cadmium stress, increases the expression of genes related to cadmium fixation and cadmium detoxification, and enhances wheat's tolerance to cadmium. These results indicate that the combined application of YG-14 and N,S-CDs helps to enrich beneficial bacteria that are tolerant to cadmium pollution, improve soil ecology, and promote plant growth, thereby improving the growth of *Sedum aizoon* in cadmium-polluted environments.

[0081] Environmental factors are important influences on rhizosphere microbial communities. Redundancy analysis (RDA) was performed on the relative abundance of soil environmental factors and various plant indicators with key differentially abundant bacterial genera to understand the impact of various environmental factors on rhizosphere microbial communities. The specific results are as follows: In the soil RDA analysis, the first and second axes explained 39.03% and 20.39% of the changes in key differentially expressed bacterial genera, respectively, with a total explained rate of 59.42%. This indicates that various soil indicators significantly affect the distribution of key differentially expressed bacterial genera. The arrows for total cadmium and acid-extractable cadmium content were the longest, indicating a close correlation between changes in bacterial genera and these factors. Specifically, the relationship is as follows: ​ and ​ It showed a negative correlation with the content of total cadmium and acid-extractable cadmium in the soil. ​ It is positively correlated with it, and in addition, ​ , ​ This is positively correlated with CEC content. ​ and ​Positive correlations were observed with organic matter content and pH. In plant RDA analysis, the first and second axes explained 48.25% and 22.74% of the changes in key differentially expressed bacterial genera, respectively, with a total explained rate of 70.99%. This indicates that various plant indicators significantly influence the distribution of key differentially expressed bacterial genera. The arrows for SOD, plant cadmium, iron, phosphorus, and sulfur content are all relatively long, indicating a close correlation between changes in bacterial genera and their distribution. Specifically, the relationships are as follows: ​ and ​ ​ It showed a negative correlation with MDA and plant cadmium content, and a positive correlation with SOD, plant iron, and phosphorus content. ​ , ​ It is positively correlated with CAT, POD and plant sulfur content. ​ It is positively correlated with the cadmium content of plants. ​ , ​ , ​ It is positively correlated with H2O2 content.

[0082] The effects of different treatments (CK group, CCK group, CDs group, YG-14 group, and Y+CDs group) on the function of rhizosphere soil bacteria in *Sedum aizoon* were investigated, and bacterial function prediction analysis was performed. The results are as follows: The enrichment of rhizosphere soil bacteria in six functional categories was shown in the KEGG primary functional taxonomy. The results showed that, compared to the CK group, the CCK, CDs, YG-14, and Y+CDs groups had increased numbers of metabolic functional genes, indicating that cadmium pollution significantly inhibited soil bacterial metabolic function. However, the application of N,S-CDs, and YG-14 helped restore the metabolic function of microorganisms affected by cadmium pollution.

[0083] The KEGG secondary functional classification heatmap shows the enrichment of rhizosphere soil bacteria in different secondary functional categories across different groups. The results indicate that compared to the CCK group, the CK group showed a decrease in genes related to Carbohydrate Metabolism, Folding, Sorting and Degradation, and Energy Metabolism. This may be because cadmium stress interferes with carbohydrate synthesis and decomposition, energy production and conversion, and leads to the accumulation of misfolded or damaged proteins within cells, affecting normal cellular function. Increased functions such as Environmental Adaptation and Circulatory System may be due to microorganisms improving their tolerance to cadmium by regulating gene expression and metabolic processes, while simultaneously enhancing the transport of nutrients and signaling molecules to cope with adversity. In conclusion, cadmium pollution severely impacts various functions of microorganisms, but they are also constantly adapting to cadmium stress. Compared to the CK group, the CDs group, YG-14 group, and Y+CDs group all showed increased membrane transport, indicating that the application of N,S-CDs and YG-14, alone or in combination, may promote microbial cell membrane transport to adapt to cadmium-polluted environments. Increased metabolism in multiple processes, such as carbohydrate metabolism and amino acid metabolism, suggests that microorganisms may have enhanced utilization of various nutrients, acquiring more energy to cope with cadmium stress by accelerating metabolism. Increased replication and repair, transcription, and other processes may indicate that microorganisms synthesize proteins involved in detoxification and repair of damaged cell structures to enhance cadmium tolerance. Increased environmental adaptation may indicate that microorganisms adjust their physiological characteristics to adapt to polluted environments. Overall, the application of N,S-CDs and YG-14, alone or in combination, can help microorganisms actively respond to cadmium stress through synergistic changes in multiple pathways, demonstrating their ability to adapt to and regulate cadmium-polluted environments.

[0084] Both the fungal dilution curve and the Shannon index curve eventually flattened out, indicating that the collected samples were accurate and reliable, and the sequencing data essentially covered all fungal communities in the soil samples from each group of Sedum sarmentosum.

[0085] The fungal Venn diagram visually presents the relationship of ASVs among the five groups. The results from the fungal Venn diagram show that the CCK group has 187 ASVs, the CK group has 143 ASVs, the CDs group has 136 ASVs, the YG-14 group has 93 ASVs, and the Y+CDs group has 81 ASVs. The CCK, CK, CDs, YG-14, and Y+CDs groups also exhibit unique characteristics. S The groups shared 107 ASVs. Similar to the results of bacterial ASV clustering analysis, the number of ASVs specific to the cadmium-free soil (CCK group) was the highest, while the number of ASVs specific to the cadmium-containing soil group was lower than that of the cadmium-free soil group, indicating that cadmium pollution affects the species richness of soil fungal communities. At the same time, there was a certain degree of sharing of fungal communities among different treatment groups, indicating that some fungal groups in the soil still have broad adaptability and survival ability in the face of cadmium pollution and different treatment factors, and can survive in a variety of soil environments.

[0086] Both the fungal PCoA and NMDS diagrams show that the CCK group was clearly separated from the other cadmium-treated groups, indicating that cadmium pollution significantly altered the soil fungal community structure. The CDs, YG-14, and Y+CDs groups were spaced apart from each other and also differed from the CK group, suggesting that the application of N,S-CDs and YG-14, alone and in combination, had different effects on the soil fungal community structure.

[0087] LEfSe analysis (LDA value > 3) of rhizosphere soil fungal communities in different treatment groups of *Sedum aizoon* revealed the following top 50 fungal groups with the most significant inter-group differences: A total of 10 differentially classified fungal groups were identified in the CCK group. Excluding unclassified groups, these were Ceratobasidiaceae, Cantharellales, ​ These groups are relatively abundant in cadmium-free soils.

[0088] A total of 12 differentially classified fungal groups were found in the CK group. Excluding unclassified groups, they were Nectriaceae, ​ , Neocosmospora_rubicola , Haematonectria , Fusarium_solani , Hypocrea_ lixii , Myrothecium_roridum , Sarocladium_strictum , Sarocladium The relative abundance of these taxa, such as Aphelidiomycota and Aphelidiomycetes, was significantly increased in the CK group, suggesting that they may have strong cadmium resistance.

[0089] A total of 7 differentially expressed fungal groups were identified in the CDs group, namely: Plectosphaerella , Plectosphaerella _cucumerina , Hypocrea_atroviridis ,Orbiliomycetes,Orbiliales,Orbiliaceae, Arthrobotrys These groups may be cadmium resistant, and N,S-CDs may contribute to their relative abundance.

[0090] One differentially expressed fungal group was found in group YG-14, which is Stachybotrys_ The unclassified form may be cadmium resistant, and YG-14 helps to increase its phase abundance.

[0091] Five differentially expressed fungal groups were identified in the Y+CDs group, namely Hypocreales, Emericellopsis , Emericellopsis_microspora Sordariomycetes, Ascomycota, and other taxa may be cadmium resistant, and the combined application of N,S-CDs and YG-14 can increase their relative abundance.

[0092] Differential species can serve as potential biomarkers to help elucidate the function and dynamics of microbial communities. Therefore, the key differential fungal species in different groups will be discussed in detail below: CCK Group Stachybotrys Plays a positive role in soil ecosystems and plant growth. Stachybotrys It is one of the dominant fungal genera in the soil microbiota, and compared with the control group, the addition of organic fertilizer significantly increased... Stachybotrys The relative abundance of [a substance] was positively correlated with the activities of soil organic carbon and β-1,4-glucosidase, and an increase in its abundance may contribute to the accumulation of soil organic carbon and nutrient cycling; D'Annibale et al. isolated [a substance] from heavily polluted aging soil. Stachybotrys sp. In the watercress germination experiment, strain DABAC 3 significantly increased the radicle length and germination index in the treatment group. This indicates that the CCK group enriches beneficial bacteria and promotes plant growth.

[0093] CK Group Neocosmospora , Haematonectria , Fusarium_solani and Myrothecium_ roridum All were harmful bacteria. These results indicate that cadmium stress leads to the accumulation of pathogens in the control group (CK group), which may have a toxic effect on the growth of *Sedum aizoon*, possibly one of the reasons for the inhibited growth of *Sedum aizoon* in the CK group. Furthermore, Hypocrea_lixii and Sarocladium These are all beneficial bacteria, for example, when applied under greenhouse conditions. Hypocrea lixii It can significantly reduce the severity of root-knot nematode disease in tomato plants, while promoting the growth of tomato plants. Sarocladium It is a genus of fungi that is tolerant to the heavy metal cadmium, selected from soil in mining areas; Sarocladiumzeae It was able to colonize wheat and effectively inhibit the development of wheat scab, reducing toxin accumulation. This indicates that the control group also accumulated some beneficial bacteria to help plants resist cadmium stress and promote their growth.

[0094] CDs group Plectosphaerella , Hypocrea_atroviridis and Arthrobotrys Both can promote plant growth and reduce plant diseases and pests. For example, Plectosphaerella plurivora SRA14 can reduce nematode damage without affecting crop growth. Hypocrea_atroviridis It has a strong antagonistic effect on soil-borne plant pathogens, inhibiting their growth in both in vitro and in vivo experiments. It also has phosphate solubility, converting insoluble phosphorus in the soil into a form that can be absorbed by plants, thus promoting plant growth. Furthermore, it has a certain ability to remove heavy metals such as cadmium and lead. Arthrobotrys It is a fungus capable of preying on nematodes, which can be used to remediate nematode-contaminated soil, improve the soil ecological environment, and produce a variety of secondary metabolites with antibacterial and antiviral effects, enhancing its application potential in biological control. The above results indicate that N,S-CDs treatment can play a role in promoting plant growth, helping plants resist diseases, and improving the soil environment by enriching beneficial bacteria.

[0095] YG-14 group Stachybotrys_ Unclassified are Ascomycota, Sordariom ycetes, Hypocreales, Hypocreales_Incertae_sedis family, Stachybotrys The species under its control have already been discussed. Stachybotrys It may play a positive role in soil ecosystems and plant growth; therefore, the application of YG-14 may promote an increase in the abundance of beneficial bacteria, thereby improving the soil environment and promoting plant growth.

[0096] Y+CDs group Emericellopsis It has the functions of promoting plant growth, improving plant resistance and improving soil ecology. Meanwhile, research shows that... Emericellopsis Natural antibacterial and antifungal compounds can be isolated, which can combat a variety of pathogens. This may enhance the plant's ability to cope with stress, thereby promoting plant growth. Emericellopsis This is a salt-tolerant fungus that can grow in high-salt environments. When used alone to treat tannery wastewater, it can significantly reduce various pollutants in the wastewater. Its purification effect is even more significant when used in combination with biochar and vetiver roots, significantly reducing the content of heavy metals such as manganese and cadmium in the wastewater. This indicates that the combined application of N,S-CDs and YG-14 enriches cadmium-tolerant beneficial bacteria, potentially enhancing the cadmium resistance of *Sedum aizoon* and promoting its growth through mechanisms such as resistance to pathogens and reduction of cadmium pollution in the soil.

[0097] Redundancy analysis (RDA) was performed on various soil and plant indicators and their relative abundance with key differentially expressed fungal genera. The results showed that the first and second axes of the soil RDA analysis explained 31.27% and 13.53% of the variation in key differentially expressed fungal genera, respectively, with a total explained rate of 44.8%. This indicates that various soil indicators significantly affect the distribution of fungal genera. Specifically, the arrows for total cadmium, acid-extractable cadmium, CEC content, and pH were all relatively long, indicating a close correlation between changes in fungal genera and their abundance. The specific relationships are as follows: Stachybotrys It showed a negative correlation with total cadmium and acid-extractable cadmium content in the soil, and a positive correlation with organic matter content and pH. Neocosmospora、Haematonectria、 Sarocladium、Arthrobotrys、Hypocrea、 Plectosphaerella It is positively correlated with the total cadmium and acid-extractable cadmium content in the soil. Emericellopsis This is positively correlated with CEC content. In the plant RDA analysis, the first and second axes explained 38.44% and 20.1% of the key differential fungal genera changes, respectively, with a total explained rate of 58.54%. This indicates that various plant indicators significantly affect the distribution of fungal genera. The arrows for POD and plant sulfur content are particularly long, indicating a close correlation between changes in fungal genera and their distribution. Specifically, the relationship is as follows: Neocosmospora、 Haematonectria、Sarocladium、Hypocrea It showed a positive correlation with MDA and H2O2 content, and a negative correlation with SOD, CAT, POD, plant cadmium, iron, phosphorus, and sulfur content. Emericellopsis Conversely, it is negatively correlated with the content of MDA and H2O2, and positively correlated with the content of SOD, CAT, POD, plant cadmium, iron, phosphorus, and sulfur. Arthrobotrys , Plectosphaerella It is positively correlated with the cadmium content of plants. Stachybotrys It is negatively correlated with the cadmium content of plants.

[0098] The effects of different treatments on the function of rhizosphere soil fungi in mineral-bearing Sedum aizoon were investigated, and fungal function prediction analysis was conducted, as detailed below: The KEGG primary functional taxonomy diagram shows the enrichment of rhizosphere soil fungi in six functional categories across different groups. The results indicate that, compared to the CK group, the CCK and Y+CDs groups showed an overall increase in the number of functional genes related to metabolism and genetic information processing, suggesting that cadmium pollution significantly inhibits multiple functions of soil fungi. However, the combined application of N,S-CDs, and YG-14 helps restore the functions of microorganisms affected by cadmium pollution.

[0099] The KEGG secondary functional classification heatmap shows the enrichment of rhizosphere soil fungi in different secondary functional categories across different groups. The results indicate that compared to the CCK group, the CK group showed reduced functions in Carbohydrate Metabolism, Energy Metabolism, Cell Growth and Death, and Transport and Catabolism. This may be because cadmium stress interferes with basic energy production pathways within organisms and hinders the synthesis and metabolism of certain substances, while also suppressing normal microbial life activities. Increased functions in Metabolism and Environmental Adaptation suggest that organisms are adjusting their metabolic pathways and physiological and biochemical characteristics to improve their tolerance to cadmium-contaminated environments.

[0100] The results above indicate that microorganisms inhibit some functions under cadmium stress, but also adapt to and resist stress by enhancing certain functions. Compared to the CK group, the functional modules in the YG-14 group all decreased. This may be because YG-14 competes with soil fungi for limited nutrients, reducing the nutrients available to the soil fungi and thus limiting their growth, thereby affecting their function. Alternatively, the metabolic activity of YG-14 in the soil may alter the soil's physicochemical properties, making the soil environment unsuitable for the survival and function of soil fungi. In contrast, most functional modules in the Y+CDs group increased. This may be because the applied N,S-CDs provide additional nutrients to the soil fungi, promoting their growth and reproduction, thus contributing to their function. Alternatively, N,S-CDs may improve the soil structure and physicochemical properties, creating a more suitable living environment for soil fungi. In conclusion, there may be a synergistic effect between YG-14 and N,S-CDs, which is more conducive to the growth and function of soil fungi.

[0101] In summary, the present invention utilizes nitrogen-sulfur-doped carbon quantum dots and endophytic bacteria to synergistically enhance the phytoremediation of heavy metal-contaminated soil. This method not only improves the absorption and translocation efficiency of heavy metals by remediation plants, breaking through the bottleneck of phytoremediation efficiency, but also enhances the stress resistance (such as antioxidant capacity) of remediation plants under cadmium stress in the soil. At the same time, it improves the rhizosphere microenvironment of plants and promotes the enrichment of beneficial microorganisms to maintain the long-term remediation effect of the soil. In other words, the remediation system constructed by the present invention has multiple effects such as promoting growth, enhancing stress resistance, and improving cadmium absorption.

[0102] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A method for synergistically strengthening phytoremediation of heavy metal contaminated soil by plant endophyte and nitrogen-sulfur doped carbon quantum dots, characterized in that, The method includes the following steps: Planting remediation plants in heavy metal-contaminated soil, applying nitrogen-sulfur-doped carbon quantum dots and endophytic bacteria to cultivate the remediation plants, and completing the remediation of heavy metal-contaminated soil.

2. The method according to claim 1, characterized in that, The plant endophytic bacteria are applied to the roots of the repaired plants via root irrigation; the amount of plant endophytic bacteria added is 2 mL applied to the roots of each repaired plant; the application frequency of the plant endophytic bacteria is once every 2 weeks.

3. The method according to claim 2, characterized in that, The plant endophytic bacteria are endophytic bacteria with heavy metal tolerance and / or plant growth-promoting functions; the remediation plant is a plant with the ability to accumulate or tolerate heavy metals.

4. The method according to claim 3, characterized in that, The plant endophytic bacteria are Enterobacter sp. YG-14; the remediation plant is Sedum sarmentosum.

5. The method according to any one of claims 1 to 4, characterized in that, The application methods of the nitrogen-sulfur-doped carbon quantum dots include at least one of the following: soil application, foliar spraying, or a combination of both.

6. The method according to claim 5, characterized in that, When applied through soil, the amount of nitrogen-sulfur-doped carbon quantum dots is 100 mg to 5000 mg per kilogram of the heavy metal-contaminated soil. When using foliar spraying, the application concentration of the nitrogen-sulfur-doped carbon quantum dots is 10 mg / L to 500 mg / L; the single application amount of the nitrogen-sulfur-doped carbon quantum dots is 1.5 mL to 5 mL; and the foliar spraying cycle of the nitrogen-sulfur-doped carbon quantum dots is 1 to 3 times per week.

7. The method according to claim 6, characterized in that, The nitrogen-sulfur-doped carbon quantum dots are prepared by hydrothermal reaction using carbon-containing precursors and nitrogen- and sulfur-containing precursors as raw materials.

8. The method according to claim 7, characterized in that, The mass ratio of the carbon-containing precursor to the nitrogen- and sulfur-containing precursor is 0.64:1.08; The carbon-containing precursor is citric acid; The nitrogen- and sulfur-containing precursor is L-cysteine; The hydrothermal reaction temperature is 160 ℃~220 ℃, and the reaction time is 4h~10h; After the hydrothermal reaction, the following treatment is also included: the product obtained after the hydrothermal reaction is subjected to ultrasonication, filtration, purification, freezing, and drying to obtain nitrogen-sulfur-doped carbon quantum dots; the ultrasonication time is 30 min to 60 min; the filtration uses a filter membrane with a pore size of 0.22 μm; the purification time is 48 h to 72 h, and the purification is carried out using a dialysis bag with a molecular weight cutoff of 500 Da; the freezing temperature is -150℃ to -80℃, and the freezing time is 12 h to 48 h; the drying is vacuum freeze-drying.

9. The method according to claim 6, characterized in that, The initial concentration of heavy metals in the contaminated soil is ≤100 mg / kg; the heavy metals in the contaminated soil include at least one of cadmium, lead, zinc, and copper.

10. The method according to any one of claims 1 to 4, characterized in that, The culture time is ≥2 weeks.

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