Method for synergistically purifying glyphosate-nitrogen and phosphorus combined pollution by inoculating piriformospora indica to strengthen wetland plants

Abstract

The invention relates to the technical field of water environment ecological restoration and pollution treatment, in particular to a method for synergistically purifying glyphosate-nitrogen and phosphorus combined pollution by inoculating piriformospora indica to strengthen wetland plants. The invention aims to provide a method for synergistically purifying glyphosate-nitrogen and phosphorus combined pollution by inoculating piriformospora indica to strengthen wetland plants, an endophytic fungus-wetland plant symbiotic system is constructed, plant growth promotion and rhizosphere micro-ecological optimization are realized, the problems of low combined pollution water treatment efficiency and poor ecological stability in the prior art are solved, and the method is suitable for industrial production. Through symbiotic system construction and experimental parameter optimization, synergistic and efficient purification of glyphosate, nitrogen and phosphorus in the aquaculture tail water is realized, and the requirements of actual aquaculture tail water environment treatment are met.

Description

Technical Field

[0001] This invention relates to the field of water environment ecological restoration and pollution control technology, and in particular to a method for inoculating Indian piriformis to enhance wetland plants and synergistically purify glyphosate-nitrogen-phosphorus compound pollution. Background Technology

[0002] Glyphosate, one of the most widely used non-selective broad-spectrum herbicides globally, is commonly used for weed control and environmental disinfection in and around aquaculture farms due to its high efficiency and low cost. It enters surrounding water bodies through various pathways, including surface runoff and aquaculture wastewater, leading to persistent and cumulative pollution. Furthermore, glyphosate's metabolite, aminomethylphosphonic acid (AMPA), is highly persistent in the environment and has complex migration and transformation pathways. It can induce oxidative stress in aquatic organisms, disrupt endocrine homeostasis, and ultimately threaten human health through bioaccumulation in the food chain, becoming a key limiting factor in the treatment of aquaculture wastewater.

[0003] Constructed wetlands, as a typical ecological wastewater treatment technology, rely on a synergistic system of "plant-substrate-microorganisms" to simultaneously remove pollutants such as nitrogen and phosphorus through multiple pathways including plant absorption, substrate adsorption, and microbial transformation. However, glyphosate, as a non-selective biotoxic substance, can inhibit the activity of 5-enolpyruvate-3-phosphate (EPSP) synthase in plants, blocking the biosynthesis of aromatic amino acids, thereby inhibiting the growth and metabolism of wetland plants, disrupting the rhizosphere microecological balance, leading to a significant decrease in nitrogen and phosphorus purification efficiency, and even plant death. Currently, the response mechanisms of wetland plants to glyphosate stress show significant interspecific differences, but related studies mostly focus on assessing the purification efficiency of single pollutants, lacking systematic research on plant tolerance screening, stress resistance mechanism analysis, and enhancement technologies under glyphosate-nitrogen and phosphorus combined pollution scenarios. This results in traditional constructed wetland systems facing technical bottlenecks such as low purification efficiency and poor ecological stability in water bodies treated by combined pollution.

[0004] As a non-host-specific endophytic fungus, *Pyrhodops indicus* exhibits broad plant symbiotic compatibility. Through multiple mechanisms, including regulating plant hormone metabolism networks, inducing the activation of plant antioxidant defense systems, and optimizing rhizosphere microbial community structure, it effectively enhances plant tolerance to abiotic stresses such as drought, salinity, and heavy metals, demonstrating promising application prospects in crop stress-resistant cultivation. However, existing research rarely addresses the symbiotic compatibility between *Pyrhodops indicus* and wetland plants, and its potential for stress enhancement and synergistic purification in glyphosate-nitrogen-phosphorus combined pollution scenarios has not yet been explored. This technological gap directly results in a long-standing lack of efficient and stable technical solutions for the ecological treatment of glyphosate-nitrogen-phosphorus combined pollution in aquaculture wastewater.

[0005] In existing technologies, traditional constructed wetland systems face challenges when dealing with glyphosate-nitrogen-phosphorus compound pollution in water bodies. These challenges include the inhibition of wetland plant growth due to glyphosate stress and the destruction of the rhizosphere microecology. Relying solely on phytoremediation technology is limited by insufficient stress resistance and low purification efficiency. Using microbial enhancement methods alone lacks host carrier support. None of these approaches can effectively overcome the synergistic bottleneck of "stress resistance + purification" in the context of glyphosate-nitrogen-phosphorus compound pollution, and cannot achieve synergistic and efficient purification of glyphosate and nitrogen-phosphorus in aquaculture water bodies.

[0006] Based on this, this invention addresses the technical needs for treating aquaculture wastewater contaminated by glyphosate and nitrogen and phosphorus. It proposes to construct a symbiotic system of Indian pyriformis and wetland plants, screen suitable wetland plants, and systematically optimize inoculation processes and purification operation parameters to simultaneously improve plant stress resistance and pollutant purification efficiency, providing a scientific basis and technical paradigm for the subsequent ecological restoration of aquaculture water bodies with compound pollution. Summary of the Invention

[0007] The purpose of this invention is to provide a method for inoculating *Pyrrosia lingua* to enhance the synergistic purification of glyphosate-nitrogen-phosphorus compound pollution in wetlands, thereby addressing the problems existing in the prior art. This invention aims to provide a method for inoculating *Pyrrosia lingua* to enhance wetland plants, constructing an endophytic fungus-wetland plant symbiotic system to promote plant growth and optimize the rhizosphere microecology. This solves the problems of low efficiency and poor ecological stability in the treatment of compound polluted water in existing technologies. Through the construction of the symbiotic system and optimization of experimental parameters, the synergistic and efficient purification of glyphosate and nitrogen-phosphorus in aquaculture wastewater is achieved, meeting the needs of actual aquaculture wastewater environmental treatment.

[0008] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for synergistic purification of glyphosate-nitrogen-phosphorus compound pollution by using Indian pyriformis to enhance wetland plants. The method includes the step of transplanting wetland plants inoculated with Indian pyriformis to the treatment site; the wetland plants include iris and / or Ruellia rubra.

[0009] Optionally, the inoculation includes the step of inoculating the roots of the wetland plant with a suspension of Indian piriformis.

[0010] Optionally, the mass percentage of *Pyrhodotorula praecox* mycelium in the *Pyrhodotorula praecox* suspension is 2%.

[0011] Optionally, the amount of the Indian piriformis suspension used is 20 mL / strain; and the inoculation is performed twice.

[0012] Optionally, when the wetland plant is Ruellia rubra, the Ruellia rubra is a Ruellia rubra cutting.

[0013] Optionally, the method for cultivating Ruellia rubra cuttings includes the step of soaking Ruellia rubra stem segments in a rooting agent before inserting them into the soil.

[0014] Optionally, the rooting agent includes naphthaleneacetic acid at a concentration of 50-300 mg / L and indolebutyric acid at a concentration of 50-300 mg / L.

[0015] Optionally, the Ruellia rubra stem segments are 8-10 cm long stem segments cut from current-year semi-lignified branches, with 2-3 plump nodes; the cutting substrate used is river sand.

[0016] Optionally, the wetland plant may be pre-cultured before inoculation with Indian piriformis.

[0017] Optionally, the pre-culture temperature is 20~25℃, the light intensity is 2000~3000 Lux, the light exposure time is 12 h, and the relative humidity is 60~70%.

[0018] The present invention discloses the following technical effects: This invention provides a method for inoculating the endophytic fungus *Pyriformis indicus* (…). Piriformospora indica This method constructs a symbiotic system of Indian pyriformis and wetland plants to enhance the tolerance of wetland plants such as iris and Ruellia rubra to glyphosate stress, and simultaneously improves their synergistic purification efficiency of nitrogen, phosphorus and glyphosate in aquaculture wastewater. In addition, this method can also improve the growth status of wetland plants and improve their various growth indicators. It is applicable to the treatment of combined pollution scenarios of agricultural non-point source pollution and aquaculture wastewater. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Schematic diagram of sand culture experimental setup for different treatments; Figure 2 Dynamic changes in DO (A), pH (B), and temperature in water bodies under different treatments. Figure 3 TN(A) and NH4 in water bodies under different treatments + -N(B), NO3 - -N(C), NO2 - -N(D) concentration dynamics and TN removal efficiency (E); Figure 4Dynamic changes in TP concentration in water under different treatments (A) and removal efficiency (B); Figure 5 Dynamic changes in COD concentration in water bodies under different treatments (A) and removal efficiency (B); Figure 6 Dynamic changes in glyphosate concentration in water under different treatments (A) and removal efficiency (B); Figure 7 The relative growth rate (A) and fresh weight growth rate (B) of plants under different treatments; In the figure, all data are expressed as Mean±SD, and different lowercase letters indicate significant differences between groups (P<0.05). Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Example 1 1. Experimental Design This experiment was conducted in a greenhouse at the Fengpu Campus Experimental Station of the Shanghai Academy of Agricultural Sciences (121°28′43″E, 30°57′15″N). The *Pyriformis indicus* involved in this experiment... Piriformospora indica ) has been disclosed in the document "Nitrogenremoval performance and mechanism in constructed wetlands under salineconditions: Role of Canna indica inoculated with Piriformospora indica In the application, the applicant promised to issue the grants for 20 years from the date of application; the wetland plants iris and Ruellia ternata were taken from the nursery of the Zhuangxing Experimental Station of the Shanghai Academy of Agricultural Sciences. The experiment was divided into the following stages: (1) Activation of Indian Pyriformis and preparation of fungal agent ① Strain activation: Using a 5 mm sterile punch, take a piece of mycelial cake from the edge of an Indian pyriformis colony, and inoculate one piece onto potato dextrose agar (PDA) medium (potato 200 g / L, glucose 20 g / L and agar 20 g / L). Place it in a constant temperature biochemical incubator at 25-28℃ and incubate in the dark for 7-10 days until Indian pyriformis forms colonies with a diameter of 5-7 cm in the PDA medium. The colonies are white and fluffy with neat edges.

[0027] ② Proliferation Culture: Take out the above-mentioned Indian piriformis culture dish, and use a sterile punch with a diameter of 5 mm to randomly select 4 mycelial blocks with a diameter of 5 mm at the edge of the mycelial growth area (avoiding the aging area in the center of the colony). Inoculate these blocks into a 500 mL Erlenmeyer flask containing 200 mL of potato broth medium (PDB, potato 200 g / L, glucose 20 g / L). Place the flask in a constant temperature shaking incubator at 25-28℃ and a rotation speed of 150-180 r / min, and culture in the dark for 5-7 days. During this period, observe the growth status of the mycelium daily until white spherical colonies of Indian piriformis mycelium are observed in the PDB medium.

[0028] ③ Preparation of Indian pyriformis agent: Collect mycelia from PDB medium, wash three times with sterile ultrapure water, add a small amount of sterile ultrapure water to a sterile tissue homogenizer and crush for 3-5 min, mix 4 g of Indian pyriformis mycelia with 200 mL of sterile ultrapure water to obtain 2% (w / v) Indian pyriformis suspension.

[0029] (2) Screening, differential treatment and pre-culture of wetland plants ① Plant selection: The irises used in the experiment were obtained from the nursery of the Zhuangxing Experimental Station of the Shanghai Academy of Agricultural Sciences (121°23′15″E, 30°53′24″N). Healthy seedlings free from pests and diseases, with a height of 15-25 cm, 4-6 leaves, undamaged fleshy roots, and no withered leaves were selected. The Ruellia ternata used in the experiment was also obtained from the nursery of the Zhuangxing Experimental Station of the Shanghai Academy of Agricultural Sciences (121°23′15″E, 30°53′24″N) and propagated through sand culture by cuttings. The specific procedure was as follows: Healthy, semi-lignified branches of the current year were selected, and 8-10 cm stem segments with 2-3 plump nodes were cut. The lower cut was made at a 45° angle, and the upper cut was made horizontally 0.5 cm from the top node. The lower leaves were removed. Clean river sand with a particle size of 0.5-2 mm was used as the cutting substrate. After rinsing three times with clean water, the sand was placed in a 121℃ high-pressure steam sterilizer for 30 minutes. After sterilization, fill the seedling trays and water thoroughly until saturated. Immerse the base 2-3 cm of the *Ruellia rutile* stem cuttings in a rooting agent mixture of 50-300 mg / L naphthaleneacetic acid (NAA, 100 mg / L in this example) + 50-300 mg / L indolebutyric acid (IBA, 100 mg / L in this example) (volume ratio 1:1) for 2 hours, stirring every 30 minutes. Use sterile bamboo sticks to make holes in the river sand of the seedling trays at 5×5 cm intervals. Insert the *Ruellia rutile* stem cuttings to a depth of 2-3 cm and compact the rooting medium. Place the trays in an intelligent seedling greenhouse, controlling the temperature at 25-28℃ and the humidity at 75-90%. Water with a 10% (v / v) Hoagland solution on days 7 and 14 after planting, at a rate of 15 mL per plant, and repeat every 20-30 minutes. Afterwards, select healthy cuttings and remove rotten or weak plants.

[0030] ② Differentiated treatment: Place the selected iris seedlings in a sterile operating table, rinse the soil particles attached to the root surface with running deionized water, gently brush the soil remaining in the crevices of the roots and the folds of the fleshy roots with a soft brush to avoid damaging the root epidermis, cut off the old, rotten roots and dead leaves with sterile scissors, rinse the roots with sterile water 3 times, and drain the water from the root surface; Place the selected Ruellia spp. seedlings in a sterile operating table, gently peel off the river sand attached to the roots with sterile tweezers to avoid damaging the newly formed fibrous roots, and cut off the old callus tissue, rotten roots and dead leaves at the base with sterile scissors.

[0031] ③ Plant pre-culture: The treated iris and Ruellia ternata seedlings were placed in 2 L seedling containers, one plant per container, and pre-cultured with 10% Hoagland solution for 2 weeks. The culture solution was changed every 3 days during this period. The seedling containers were placed in an artificial climate chamber with the following conditions: temperature 20~25℃, light intensity 2000~3000 Lux, light-dark ratio of 12 h:12 h (12 h of light time and 12 h of darkness), and relative humidity of 60~70%. The growth status of the plants was observed daily, and newly formed dead leaves were removed in time. After the pre-culture, plants with green leaves, no pests or diseases, and good root growth were selected for subsequent inoculation with Pyriformis indica.

[0032] (3) Construction of the Indian piriformis-wetland plant symbiotic system ① Inoculation with *Pyrrosia lingua*: Place 50% of the selected healthy iris and *Ruellia rutile* seedlings in a 1 L seedling container, one seedling per container, and add 300 mL of 10% (v / v) Hoagland solution to ensure that the roots are fully in contact with the culture solution. On the 3rd and 7th day of hydroponic cultivation of the healthy iris and *Ruellia rutile* seedlings, inject 20 mL of 2% (w / v) *Pyrrosia lingua* suspension into the dense root zone of each seedling. After each inoculation, gently shake the seedling container to ensure that the *Pyrrosia lingua* suspension is evenly distributed in the culture solution and that all parts of the plant roots are fully in contact with the inoculant.

[0033] ② Colonization detection of *Pyrhodotorula praecox*: On day 7 post-inoculation, three *Iris* and *Ruellia rutile* plants were randomly selected. The root surface was rinsed with deionized water, and the roots were cut into 3-5 mm segments and soaked in 10 wt% NaOH for 4 hours. The roots were then rinsed three times with deionized water, soaked in 1 wt% hydrochloric acid for 5 minutes, and rinsed with water to remove surface residue. The roots were then stained with 0.05 wt% tylosin blue in the dark for 15 minutes. The staining residue was washed away with deionized water until the background color faded. After blotting with filter paper, the presence of *Pyrhodotorula praecox* hyphae or chlamydospores was observed using a Leica DM2000 microscope at 10×40 magnification. The results showed that *Pyrhodotorula praecox* successfully colonized *Iris* and *Ruellia rutile*.

[0034] ③ Experimental system construction: Select a 5 L cylindrical cultivation container, make an opening 2-3 cm from the bottom of the container and equip it with a 6-point water tap. Use 0.5-2 mm clean river sand as the cultivation substrate. After rinsing with clean water 3 times, place it in a 121℃ high-pressure steam sterilizer for 30 min. After cooling to room temperature, fill the 5 L cultivation container with river sand to a height of 10 cm. After filling, water it thoroughly with sterile water and let it stand for 24 h to drain excess water. After inoculating the seedlings with Indian piriformis, remove the iris and Ruellia ternata seedlings from the seedling trays, rinse the roots with sterile water to remove any residual culture solution, and transplant them into 5 L cultivation containers filled with 10 cm of river sand. Transplant one seedling per container, ensuring that the roots are naturally spread out during transplanting. Cover the roots with river sand until they are completely buried, and gently compact the sand to secure the plant. Place a 15×10 cm 100-mesh nylon mesh bag inside the drainage outlet of the cultivation container, with 3-5 mm glass beads inside, filling the bag to 70-80% capacity. Ensure that the bag fits tightly against the drainage hole to ensure that water can only flow out after being filtered through the mesh bag, preventing the river sand substrate from overflowing with the water. After transplanting, the plants were cultured in 10% (v / v) Hoagland solution, and the experiment was started after 2 weeks. The treatments of Iris tectorum and Ruellia rutile were designated as I-Pi (Iris + Iris tectorum inoculation group) and R-Pi (Ruellia rutile + Iris tectorum inoculation group), respectively. Meanwhile, Iris and Ruellia rutile seedlings that were not inoculated with Iris tectorum were used as control treatments and designated as I-CK (Iris control group) and R-CK (Ruellia rutile control group), respectively.

[0035] Tap water was placed in an open container and left to stand under natural sunlight for 5 days to remove residual chlorine. Then, ammonium chloride (NH4Cl), potassium nitrate (KNO3), potassium dihydrogen phosphate (KH2PO4), and glucose (C6H2PO4) were added to the treated water. 12 Artificial wastewater was prepared using O6 and glyphosate (C3H8NO5P) with the following concentrations: total nitrogen (TN) 16.01±0.22 mg / L, total phosphorus (TP) 2.75±0.43 mg / L, chemical oxygen demand (COD) 52.67±4.04 mg / L, and glyphosate (C3H8NO5P) 2328.13±173.97 μg / L.

[0036] During the experiment, a static mode was used. After a single water intake, water samples were collected every 2 days, stored in ice boxes, and brought back to the laboratory for analysis. The water quality indicators were determined as follows: water temperature, dissolved oxygen (DO), and pH were measured in situ using an HI9829 (HANNA, Italy); total nitrogen (TN) was determined using alkaline potassium persulfate oxidation-UV spectrophotometry; and NH4 was determined using a flow injection automated analyzer (Seal, AA3, Germany). + -N, NO3 - -N and NO2 --N concentration; total phosphorus (TP) in water was determined by ammonium molybdate spectrophotometry; glyphosate (C3H8NO5P) was determined by ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (AB, USA).

[0037] 2. Experimental Results This invention uses simulated aquaculture wastewater as the treatment object and constructs four experimental systems (such as...). Figure 1 As shown in the figure, they are (1) I-CK (Iris control group); (2) I-Pi (Iris + inoculated with Indian piriformis treatment group); (3) R-CK (Ruellia control group); (4) R-Pi (Ruellia + inoculated with Indian piriformis treatment group).

[0038] The experiment lasted 16 days, with water samples collected every 2 days. Changes in water physicochemical parameters, nitrogen, phosphorus, COD, and glyphosate concentrations and removal rates were measured. At the end of the experiment, the fresh weight of the plants was measured. The results are as follows: (1) During the experiment, DO showed a trend of first rapidly decreasing and then increasing. Figure 2 (A) This is because in the early stage of the experiment (day 0-day 2), due to the influence of plant root respiration and the oxygen consumption process of microbial decomposition of organic pollutants, the DO values ​​of each group reached their lowest value on day 2. The DO values ​​of the I-CK, I-Pi, R-CK, and R-Pi groups were 0.98±0.41, 1.09±0.38, 1.86±0.71, and 1.77±0.83 mg / L, respectively. By the end of the experiment, there was no significant difference among the groups (P>0.05). From day 2 onwards, the DO values ​​of the Iris treatment inoculated with Indian pyriformis (I-Pi) were slightly lower than those of the uninoculated group (I-CK), while the DO values ​​of the Ruellia treatment inoculated with Indian pyriformis (R-Pi) were slightly higher than those of the uninoculated group (R-CK). (2) The pH of each group showed a trend of first decreasing and then slowly increasing. Figure 2 In (B) of the experiment, the pH value reached its lowest point in the early stage (day 2). By the end of the experiment, the pH values ​​of the I-CK, I-Pi, R-CK, and R-Pi groups were 7.64±0.07, 7.63±0.04, 7.86±0.01, and 7.70±0.07, respectively. Through inter-group comparison, it can be seen that the pH of the Ruellia rubra group (R-CK, R-Pi) was generally higher than that of the Iris group (I-CK, I-Pi), reflecting the difference in the influence of different plant types on the pH of the system. However, the pH difference between the control group (CK) and the Pi treatment group (Pi) of the same species was very small, indicating that the inoculation of Pyriformis in India had no significant effect on the pH of the system. (3) The water temperature in each group showed a trend of first rising and then falling, then rising again and then falling rapidly. Figure 2 (C) During the entire experiment, the water temperature was 16.54±4.50℃, and there was no significant difference between the groups; (4) The TN concentration in the four treatment groups showed a continuous decreasing trend over the experimental time. Figure 3 The concentrations of TN in the *Iris* group (I-CK, I-Pi) decreased from 16.01 ± 0.22 mg / L on day 0 to 2.13 mg / L on day 16, showing that both plants significantly removed TN from the system under glyphosate stress. Before day 10, the TN concentration in the *Iris* group (I-CK, I-Pi) decreased at a faster rate than that in the *Ruellia ternata* group (R-CK, R-Pi). In the later stages of the experiment (days 10 to 16), the TN concentration in the *Pyrrosia lingua* inoculated group (I-Pi, R-Pi) decreased at a faster rate than that in the uninoculated group (I-CK, R-CK), with R-Pi showing the fastest decrease. (5) By the end of the experiment, the average TN removal rate of each group was R-Pi (86.69%) > I-Pi (74.68%) > R-CK (68.64%) > I-CK (64.80%). Figure 3 (E)). Under the same plant, the removal rate of "Indian pyriformis inoculated group (Pi)" was higher than that of "control group (CK)" (R-Pi>R-CK, I-Pi>I-CK). The average TN removal rate of Iris + Indian pyriformis inoculated group (I-Pi) was 9.88% higher than that of I-CK, and that of Ruellia + Indian pyriformis inoculated group (R-Pi) was 18.05% higher than that of R-CK. This indicates that Indian pyriformis inoculation can further improve the TN removal effect. At the same time, the removal rate of the R-Pi group was higher than that of other groups, indicating that Indian pyriformis inoculation has a more significant promoting effect on TN removal in Ruellia.

[0039] (6) 4 groups of NH4 treatment + -N concentration showed a continuous decreasing trend with experimental time. Figure 3 In (B) of the study, at day 10, the concentration of R-Pi in the R-Pi group decreased to 0.114 mg / L, approaching 0 mg / L. Under the same plant conditions, the concentration of Iris + inoculated with *Pyrrosia lingua* (I-Pi) was slightly higher than that of its control group (I-CK), while the concentration of Ruellia rutile + inoculated with *Pyrrosia lingua* (R-Pi) was slightly lower than that of the Iris control group (I-CK). This indicates that inoculation with *Pyrrosia lingua* increases NH4 levels in Ruellia rutile. + It has a significant promoting effect on NH4+ removal efficiency, while on iris NH4+ removal efficiency. + -N removal had no significant gain effect; in the later stages of the experiment (days 10-16), the overall NH4+ in the *Ruellia ternata* group was significantly reduced. + -N concentrations were generally lower than in the Iris group, indicating that *Ruellia ternata* has a lower NH4+ concentration than *Ruellia ternata*. + -N has better removal efficiency.

[0040] (7) 4 groups of NO3 treatment- -N concentration showed an increasing and then decreasing trend with experimental time. Figure 3 In the (C) group, the concentration of NO3- showed a significant jump from day 10 to 12. The peak value was most prominent in the *Ruellia ternata* inoculation group (R-Pi), reaching 6.92 ± 0.45 mg / L on day 10. The peak value in the *Ruellia ternata* control group (R-CK) rose synchronously to 6.54 ± 0.49 mg / L. However, the peak values ​​in the *Iris* groups (I-CK, I-Pi) were delayed and lower than those in the *Ruellia ternata* groups (R-CK, R-Pi), rising to 5.23 ± 0.37 mg / L and 4.13 ± 1.26 mg / L respectively on day 12. Regarding inter-group characteristics, the species difference was mainly in NO3-. - The core influencing factors of NO3- concentration fluctuations were: the concentration fluctuation amplitude of the *Ruellia ternata* group (R-CK, R-Pi) was significantly greater than that of the *Iris* group (I-CK, I-Pi), and the "peak height" and "later decline amplitude" of R-Pi were the most significant; by the end of the experiment, the four groups treated with NO3- - The average concentrations of NO3- were I-CK (5.17 mg / L) > R-CK (5.02 mg / L) > I-Pi (3.85 mg / L) > R-Pi (2.07 mg / L), indicating that inoculation with *Pyriformis indica* effectively enhanced the NO3- uptake of both plants. - The removal efficiency of -N was improved, with a more significant synergistic effect on Ruellia ternata.

[0041] (8) 4 groups of NO2 treatment - -N concentration showed an increasing and then decreasing trend with experimental time. Figure 3 (D) in the middle), until the end of the experiment, the NO2 in each group - The average NO2 concentrations were as follows: I-CK (0.58 mg / L) > I-Pi (0.23 mg / L) > R-CK (0.08 mg / L) > R-Pi (0.01 mg / L). Under the same plant treatment, the NO2 concentrations in the inoculated groups (I-Pi, R-Pi) were... - -N residue levels were lower than in the control groups (I-CK, R-CK), indicating that inoculation with *Pyridis praecox* has a lower NO2 content than the control groups. - The removal of -N also had a significant promoting effect, while NO2 in the *Ruellia ternata* group was reduced. - The final NO2 concentration was lower than that of the Iris group, indicating that the combination of "Ruellia ternata + Pyridis praecox" reduced NO2. - It performs best in terms of -N accumulation and can effectively reduce NO2 during nitrogen conversion. - -N intermediate residual risk.

[0042] (9) The TP concentration in the four treatment groups showed a continuous decreasing trend over time. At the end of the experiment, the average TP concentration in each group was: I-CK (1.01 mg / L) > R-CK (0.67 mg / L) > I-Pi (0.56 mg / L) >> R-Pi (0.35 mg / L). Figure 4 In (A), the average TP removal rate for each group was R-Pi (87.23%) > I-Pi (79.76%) > R-CK (75.50%) > I-CK (63.33%). Figure 4 In (B) of the study, under the same plant, the removal rate of the "Inonotus indicus treatment group (Pi)" was higher than that of the "control group (CK)" (R-Pi>R-CK, I-Pi>I-CK) and significantly improved the plant's removal effect on TP (P<0.05). Among them, the average TP removal rate of Iris + Inonotus indicus treatment group (I-Pi) was 16.43% higher than that of I-CK, and the 'Ruellia scabra' + Inonotus indicus treatment group (R-Pi) was 11.73% higher than that of R-CK.

[0043] (10) The COD concentrations in the four treatment groups showed a rapid decrease followed by a gradual decline. At the end of the experiment, the average COD concentrations in each group were: R-CK (11.00 mg / L) > I-CK (9.67 mg / L) > R-Pi (8.67 mg / L) > I-Pi (6.33 mg / L). Figure 5 In (A), the average COD removal rate for each group was I-Pi (87.97%) > R-Pi (83.54%) > I-CK (81.64%) > R-CK (79.11%). Figure 5 In (B) of the study, there were no significant differences among the groups (P>0.05). Under the same plant, the removal rate of the "Inonotus inoculated group (Pi)" was higher than that of the "control group (CK)" (R-Pi>R-CK, I-Pi>I-CK). The average COD removal rate of Iris + Inonotus inoculated group (I-Pi) was 6.33% higher than that of I-CK, and that of Ruellia + Inonotus inoculated group (R-Pi) was 4.43% higher than that of R-CK. This indicates that Inonotus inoculation can effectively improve the COD removal effect.

[0044] (11) The glyphosate concentration in the four treatment groups showed a continuous decreasing trend. By the end of the experiment, the average glyphosate concentration was R-CK (594.83 μg / L) > I-CK (445.20 μg / L) > R-Pi (434.20 μg / L) > I-Pi (147.75 μg / L). Figure 6 In (A) of the group, the average glyphosate removal rate was I-Pi (93.65%) > R-Pi (81.35%) > I-CK (80.99%) > R-CK (74.45%). Figure 6 In (B) of the study, for the same plant, inoculation with Indian pyriformis significantly improved the glyphosate removal effect (P>0.05). In the iris group, the removal rate of I-Pi was 12.66% higher than that of I-CK, and in the Ruellia rubra group, the removal rate of R-Pi was 6.90% higher than that of R-CK.

[0045] (12) By the end of the experiment, the relative growth rates of the four treatments were ranked as R-Pi (0.0186 g·g⁻¹). -1 ·d -1 R-CK (0.0156 g·g) > R-CK (0.0156 g·g) -1 ·d -1 )>I-Pi (0.0077 g·g -1 ·d -1 >I-CK (0.0058 g·g) -1 ·d -1 () Figure 7 In (A) of the data, the fresh weight growth rate was ranked as follows: R-Pi (34.78%) > R-CK (28.32%) > I-Pi (13.16%) > I-CK (9.79%). Figure 7 In (B) of the study, both growth indicators of the *Ruellia rutabaga* group (R-CK, R-Pi) were significantly higher than those of the *Iris* group (I-CK, I-Pi), indicating that species characteristics are the dominant factor regulating plant growth. For the same plant, the growth indicators of the treatment groups inoculated with *Pyrrosia lingua* (R-Pi, I-Pi) were slightly higher than those of the uninoculated control group, indicating that *Pyrrosia lingua* inoculation had a certain promoting effect on the relative growth rate and fresh weight growth rate of the two plants, but it was not significant (P>0.05).

[0046] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for synergistically purifying glyphosate-nitrogen-phosphorus combined pollution by using G. indica to strengthen wetland plants, characterized in that, The method comprises the step of transplanting wetland plants inoculated with Magnaporthe grisea to the land to be treated; the wetland plants comprise Iris and / or Acorus calamus.

2. The method of claim 1, wherein, The inoculation comprises the step of inoculating a Magnaporthe grisea suspension to the root system of the wetland plants.

3. The method of claim 2, wherein, The mass percentage of Magnaporthe grisea mycelium in the Magnaporthe grisea suspension is 2%.

4. The method of claim 2, wherein, The amount of the Magnaporthe grisea suspension used is 20 mL per plant; the number of inoculation times is 2.

5. The method of claim 2, wherein, When the wetland plants are Acorus calamus, the Acorus calamus are Acorus calamus cutting seedlings.

6. The method of claim 5, wherein, The culture method of the Acorus calamus cutting seedlings comprises the step of soaking Acorus calamus stems in a rooting agent and then cutting.

7. The method of claim 6, wherein, The rooting agent comprises naphthalene acetic acid with a concentration of 50-300 mg / L and indole butyric acid with a concentration of 50-300 mg / L.

8. The method of claim 6, wherein, The Acorus calamus stems are 8-10 cm stem segments of current-year semi-lignified branches with 2-3 full nodes; the cutting substrate used for cutting is river sand.

9. The method of claim 1, wherein, Before the inoculation of Magnaporthe grisea, the wetland plants are pre-cultured.

10. The method of claim 9, wherein, The pre-culture temperature is 20-25℃, the light intensity is 2000-3000 Lux, the light time is 12 h, and the air relative humidity is 60-70%.

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