A method for rapid screening and comprehensive evaluation of salt-tolerant submerged plants in estuary

Abstract

This invention relates to the field of screening and application of salt-tolerant submerged plants, and particularly to a rapid screening and comprehensive evaluation method for salt-tolerant submerged plants in estuaries. This method includes the following steps: Step 1, placing the submerged plants to be screened under artificially simulated salt stress conditions with different salinity gradients for cultivation, and recording the survival rate, growth indicators, and physiological indicators of the plants; Step 2, determining the salt tolerance survival threshold of each plant based on the survival rate and growth indicators; Step 3, analyzing the physiological response characteristics of each plant under salt stress based on physiological indicators; Step 4, selecting plants with strong salt tolerance and further measuring their removal efficiency of nitrogen and phosphorus nutrients in the water; Step 5, comprehensively considering salt tolerance and nutrient removal capacity, screening out the submerged plants with the best overall performance. The method described in this invention is simple to operate, has a short cycle, and provides comprehensive evaluation indicators, possessing strong practicality and promotional value.

Description

Technical Field

[0001] This invention relates to the field of screening and application of salt-tolerant submerged plants, and in particular to a rapid screening and comprehensive evaluation method for salt-tolerant submerged plants in estuaries. Background Technology

[0002] Coastal wetlands, estuaries, and brackish water areas are affected by both natural hydrological conditions and human activities, generally facing environmental problems such as increased salinity fluctuations and nitrogen and phosphorus nutrient enrichment. Eutrophication is a significant trend, severely threatening the structure and function of aquatic ecosystems. Submerged plants, as key primary producers in aquatic ecosystems, not only stabilize water sediment, inhibit algal blooms, and maintain clear water homeostasis, but also efficiently remove nutrients such as nitrogen and phosphorus from water through various pathways including absorption, adsorption, enrichment, and rhizosphere microenvironment regulation. They are among the most widely used and stable biological materials for aquatic ecological restoration and water purification, possessing significant advantages such as high ecological safety, low restoration costs, and good landscape effects. Most submerged plants are adapted to freshwater habitats and are sensitive to salt stress. In environments with increased salinity, they are prone to osmotic imbalance, reduced photosynthetic efficiency, and inhibited growth, leading to decreased survival and water purification capacity. Therefore, a systematic evaluation of the salt tolerance and nutrient removal capacity of common submerged plants under simulated salt stress conditions, and the screening of superior species with both strong salt tolerance potential and high nitrogen and phosphorus removal efficiency, is of great significance for improving the stability and practicality of ecological restoration in brackish water interaction areas. Summary of the Invention

[0003] Based on the above, this invention provides a rapid screening and comprehensive evaluation method for salt-tolerant submerged plants in estuaries. Using various common submerged plants as experimental materials, this invention compares their salt tolerance and growth adaptability under simulated salt stress conditions, initially screening for dominant salt-tolerant species. Further, through static simulation experiments, it explores the removal capacity and purification characteristics of the selected species for nitrogen and phosphorus nutrients in the water, comprehensively evaluating their salt tolerance and water purification potential. Finally, it identifies the submerged plant with the best overall performance as the material for subsequent experiments, laying a species-based foundation for in-depth research on its salt tolerance physiological and molecular mechanisms and its response to complex stresses.

[0004] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is a method for rapid screening and comprehensive evaluation of salt-tolerant submerged plants in estuaries, comprising the following steps: Step 1: The submerged plants to be screened were cultured under artificially simulated salt stress conditions with different salinity gradients, and the survival rate, growth indicators and physiological indicators of the plants were recorded. Step 2: Determine the salt tolerance survival threshold for each plant based on its survival rate and growth indicators; Step 3: Analyze the physiological response characteristics of each plant under salt stress based on physiological indicators; Step 4: Select plants with strong salt tolerance and further determine their removal efficiency of nitrogen and phosphorus nutrients in water. Step 5: Based on the combination of salt tolerance and nutrient removal capacity, the submerged plants with the best overall performance are selected.

[0005] In a preferred embodiment of the present invention, in step 1, the salinity gradient is 0%, 0.5%, 1.0%, and 2.0%; the cultivation conditions are set as follows: temperature 25±2℃, light intensity 4000 lux, light-dark cycle 12h / 12h, and cultivation period 14 days; the submerged plants include at least one of Vallisneria natans, Elodea nuttallii, Potamogeton pectinatus, Hydrilla verticillata, Ceratophyllum demersum, and Myriophyllum spicatum.

[0006] In a preferred embodiment of the present invention, in step 1, the survival rate is determined by the following criteria: complete loss of chlorosis of the plant leaves and softening and lodging of the stems are used as the basis for determining mortality; the growth indicators include plant height and relative water content of leaves; the physiological indicators include soluble protein content, relative conductivity and total chlorophyll content of leaves.

[0007] In a preferred embodiment of the present invention, in step 2, the criterion for determining the salt tolerance survival threshold is: the survival rate of the plant after 14 days of cultivation at a certain salinity is ≥50% as the basis for its ability to tolerate that salinity.

[0008] In a preferred embodiment of the present invention, step 3, the analysis of the physiological response characteristics includes: comparing the changes in the soluble protein content of leaves of various plants under different salinity treatments, the degree of damage to cell membrane integrity, and the total chlorophyll content; the cell membrane integrity is characterized by relative conductivity.

[0009] In a preferred embodiment of the present invention, in step 4, the method for determining the nitrogen and phosphorus nutrient removal efficiency is as follows: in a static simulation experiment, the dynamic changes of ammonia nitrogen and total phosphorus concentrations in the water under different salinity conditions are monitored, and the ammonia nitrogen removal rate and total phosphorus removal rate within a 14-day culture period are calculated.

[0010] In a preferred embodiment of the present invention, the submerged plant with the best overall performance in step 5 is *Myriophyllum spicatum*. It exhibits high survival rate and stable physiological indicators under high salt stress, and its removal efficiency for ammonia nitrogen and total phosphorus is superior to other tested plants.

[0011] The second technical solution of this invention is a method for screening salt-tolerant submerged plants suitable for ecological restoration of coastal wetlands, estuaries, or brackish water areas, using the aforementioned rapid screening and comprehensive evaluation method for salt-tolerant submerged plants in estuaries.

[0012] Compared with the prior art, the present invention has the following beneficial effects: 1. A systematic salt tolerance screening system was constructed: This invention establishes a scientific and systematic screening method for salt-tolerant submerged plants by setting up multi-gradient salinity (0%, 0.5%, 1.0%, 2.0%) stress experiments and combining multi-dimensional evaluation parameters such as survival rate, growth indicators (plant height, relative water content), and physiological indicators (soluble protein content, relative conductivity, chlorophyll content). This method can quickly and accurately assess the response characteristics and tolerance thresholds of different submerged plants to salt stress.

[0013] 2. Achieving a comprehensive evaluation of salt tolerance and water purification capacity: Based on salt tolerance screening, this invention further conducts experiments on nitrogen and phosphorus nutrient removal capacity, combining the salt tolerance physiological characteristics of plants with ecological restoration functions (ammonia nitrogen and total phosphorus removal rates) to comprehensively evaluate the salt tolerance adaptability and water purification potential of plants. This overcomes the limitations of traditional research that only focuses on a single indicator, and provides a more practical basis for species selection for the ecological restoration of brackish water areas.

[0014] 3. Clarified key salt tolerance thresholds and species differences: This invention systematically reveals for the first time that 1.0% salinity is the key threshold for distinguishing salt-tolerant species from sensitive species. It clarifies the survival rate, growth inhibition degree, and physiological response differences of six common submerged plants (Vallisneria natans, Elodea nuttallii, Potamogeton pectinatus, Hydrilla verticillata, Ceratophyllum demersum, and Myriophyllum sp.) under different salinities. It identifies Potamogeton pectinatus and Myriophyllum sp. as high-salt-tolerant species, laying a species basis for subsequent physiological and molecular mechanism research and complex stress response analysis.

[0015] 4. Screening out superior species with both salt tolerance and high purification efficiency: Through comprehensive evaluation of multiple indicators, this invention found that *Myriophyllum spicatum* not only has strong salt tolerance (survival rate >50% at 2.0% salinity, relative conductivity <50%, and highest chlorophyll content), but also performs excellently in the removal of ammonia nitrogen and total phosphorus (removal rate higher than *Potamogeton pectinata* at all salinity gradients). It is an ideal species with both salt tolerance and high water purification efficiency, and is suitable for ecological restoration projects in nearshore brackish water confluence areas.

[0016] 5. High application and promotion value: The method described in this invention is simple to operate, has a short cycle, and comprehensive evaluation indicators. It can be widely applied to the screening of salt-tolerant submerged plants and the selection of ecological restoration species in coastal wetlands, estuaries, and brackish water areas. It has strong practicality and promotion value, and provides scientific support and technical guarantee for improving the stability and restoration effect of such fragile ecosystems. Attached Figure Description

[0017] 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.

[0018] Figure 1 This invention relates to an experimental apparatus for submerged plants.

[0019] Figure 2 Protein content of plant leaves on day 14: (a) 0% salinity, (b) 0.5% salinity, (c) 1.0% salinity, (d) 2.0% salinity, (e) different plant types; In the figure: "*" indicates a significant difference between different salt treatment groups and the control group (0% salinity) for the same plant, "*" indicates p < 0.05, and "**" indicates p < 0.01.

[0020] Figure 3 The relative electrical conductivity of plant leaves on day 14 is: (a) 0% salinity, (b) 0.5% salinity, (c) 1.0% salinity, (d) 2.0% salinity, (e) different plant types; In the figure, "*" indicates a significant difference between different salt treatment groups and the control group for the same plant, "*" indicates p < 0.05, and "**" indicates p < 0.01.

[0021] Figure 4 The total chlorophyll content of plant leaves on day 14 is: (a) 0% salinity, (b) 0.5% salinity, (c) 1.0% salinity, (d) 2.0% salinity, (e) different plant types; In the figure, "*" indicates a significant difference between different salt treatment groups and the control group for the same plant, "*" indicates p < 0.05, and "**" indicates p < 0.01.

[0022] Figure 5 Changes in ammonia nitrogen: (a) Potamogeton crispus, (b) Myriophyllum spicatum, (c) Total removal rate.

[0023] Figure 6 Total phosphorus removal: (a) Potamogeton crispus, (b) Myriophyllum spicatum, (c) Total removal rate. Detailed Implementation

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field. Unless otherwise specified, the reagents or raw materials used are all purchased from commercial channels or are publicly available. Unless otherwise specified, all reagents are of analytical grade.

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1 1. Materials and Methods 1.1 Test Materials Based on preliminary surveys and analyses of plants in coastal areas surrounding Guangdong, and in conjunction with commonly used species for estuarine bioremediation, *Vallisneria natans* was selected. Vallisneria natans Elodea ( Elodea nuttallii ), Potamogeton crispus ( Potamogeton pectinatus ), Elodea ( Hydrilla verticillata ), goldfish algae ( Ceratophyllum demersum ), Myriophyllum spicatum ( Myriophyllum spicatumSix submerged plant species were used as experimental materials. All plants were purchased from an aquatic product store in Honghu City, Hubei Province. After cleaning and pruning, the plants were pre-cultured in a 1 / 2 concentration modified Hoagland nutrient solution for 7-10 days, with the nutrient solution changed every 2 days. The culture conditions were a 12-hour light / 12-hour dark cycle, a temperature of 25±2℃, and a light intensity of 4000 lux. After the plants recovered and stabilized, individuals with uniform growth were selected for subsequent experiments.

[0032] 1.2 Main Experimental Reagents The experimental reagents were mainly used for the pre-culture of six submerged plants and the measurement of physiological and water quality indicators. The experimental reagents are shown in Table 1. Hoagland nutrient solution formula: K2SO4 607 mg / L, NH4H2PO4 115 mg / L, MgSO4 493 mg / L, EDTA sodium iron salt 20 mg / L, FeSO4 2.86 mg / L, borax 4.5 mg / L, MnSO4 2.13 mg / L, CuSO4 0.05 mg / L, ZnSO4 0.22 mg / L, (NH4)2SO4 0.02 mg / L.

[0033] Table 1 Main reagents for the experiment

[0034] 1.3 Main Instruments and Equipment The main experimental instruments and equipment used are shown in Table 2.

[0035] Table 2 Experimental Instruments and Equipment

[0036] 1.4 Experimental Setup Experimental apparatus (such as) Figure 1 The container shown is a cylindrical acrylic glass container, 400 mm high, 150 mm wide, and 3 mm thick. Each container contains 5 L of experimental water and 30 mm of quartz sand (2-7 mm, Shanghai Aladdin Biochemical Technology Co., Ltd.). The submerged plants were cultured in a controlled laboratory environment simulating natural conditions, with a 12 h light / 12 h dark cycle, a temperature of 25 ± 2 °C, and a light intensity of 4000 lux.

[0037] 1.5 Experimental Design 1.5.1 Survival rate experiment of submerged plants under salt stress Six submerged plant species with consistent growth after pre-culturing were selected as experimental materials. Salt solutions with salinities of 0% (control), 0.5%, 1.0%, and 2.0% were prepared using NaCl and Hoagland nutrient solution. Five plants were transplanted into cylindrical containers containing 5L of experimental water, with three biological replicates per treatment. The experiment was conducted under controlled conditions: a 12h light / 12h dark cycle, a temperature of 25±2℃, and a light intensity of 4000 lux. The experimental period was 14 days. During the experiment, water salinity was monitored daily, and deionized water was added to maintain salinity stability. Plant growth was observed and recorded daily, with complete leaf chlorosis and stem softening / collapse as the mortality criteria. The survival rate of each treatment group was calculated to determine their salt tolerance threshold. Furthermore, plant height and relative leaf water content were measured on days 7 and 14 to analyze the effects of salt stress on plant growth and water status.

[0038] 1.5.2 Experiment on the physiological response of submerged plants under salt stress Six submerged plant species with consistent growth after pre-culturing were selected and transplanted into containers containing 5L of experimental water, with 5 plants per container. Three biological replicates were set up for each treatment. Salt solutions with salinities of 0% (control), 0.5%, 1.0%, and 2.0% were prepared using NaCl and Hoagland nutrient solution. The experimental period was 14 days. Salinity was monitored daily and maintained at a stable level throughout the experiment, with culture conditions consistent with the survival rate experiment. At the end of the experiment (day 14), leaves were collected from the same parts of the plants in each treatment group. After rinsing with distilled water and drying with filter paper, the leaves were weighed, and three physiological indicators—protein content, relative conductivity, and total chlorophyll—were measured. All indicators were measured in triplicate, and the average values ​​were used for statistical analysis.

[0039] The formula for calculating the relative height of each plant is as follows: (1-1) X t X represents the average plant height (cm) of the treatment group at time t, X0 represents the average plant height (cm) of the control group at time t, and Y represents the relative plant height (%).

[0040] The formula for calculating the relative water content (RWC) of leaves of each plant is as follows: (1-2) RWC represents the relative water content of the leaf, W f W represents the fresh weight (g) of the leaves. d W represents the dry weight (g) of the leaf. t This indicates the weight (g) of the leaf tissue after it has been fully saturated with water.

[0041] The formula for calculating the relative electrical conductivity (%) of each plant leaf is as follows: (1-3) R represents the relative electrical conductivity (%) of the plant leaves. The electrical conductivity of the sample leaves was measured after soaking them in deionized water for 12 hours, which was R1 (S / m). The electrical conductivity of the sample leaves was measured after cooling them in a boiling water bath for 30 minutes, which was R2 (S / m).

[0042] The formula for calculating the chlorophyll concentration in the leaves of each plant is as follows: (1-4) (1-5) (1-6) D 663 D is the absorbance at a wavelength of 663 nm. 645 The absorbance is measured at a wavelength of 645 nm. Chl a and Chl b are the concentrations (mg / L) of chlorophyll a and chlorophyll b, respectively, and Chl is the total chlorophyll concentration (mg / L).

[0043] The formula for calculating the soluble protein content is as follows: (1-7) 1.5.3 Experiment on the nitrogen and phosphorus removal effect of submerged plants under salt stress Following preliminary screening, salt-tolerant submerged plants were selected for nitrogen and phosphorus nutrient removal experiments. Plants with uniform growth were chosen for the experiment, washed with tap water, and then transplanted into containers containing 5L of experimental water. Five plants were planted per container, secured with plastic planting baskets and planting cotton. The experiment included four salinity gradients: 0%, 0.5%, 1.0%, and 2.0%. NaCl was used to adjust the salinity. The experimental water was artificially prepared eutrophic water. Nessler's reagent method and molybdenum blue colorimetric method were used to determine the ammonia nitrogen and total phosphorus content in the water. The experiment lasted 14 days. The water quality indicators and corresponding reagent dosages are as follows: Table 3 Water quality for experiments

[0044] Formula for calculating the nutrient removal rate (%) of plants in water: (1-8) R represents the removal percentage (%), C0 represents the initial concentration (mg / L), and C t This represents the concentration (mg / L) at time t.

[0045] 2 Results 2.1 Assessment of the survival threshold of submerged plants under salt stress 2.1.1 Plant survival status Six plant species, namely Vallisneria natans, Elodea nuttallii, Potamogeton crispus, Hydrilla verticillata, Ceratophyllum demersum, and Myriophyllum sp., were selected for salt stress experiments, and the plant survival rates were obtained (as shown in Table 4).

[0046] Table 4. Survival rates of six submerged plants under different salinity treatments

[0047] Note: "++" indicates that all hydroponic plants survive normally; "++-" indicates that some hydroponic plants wither and die, with a survival rate of 50-100%; "--" indicates that most hydroponic plants wither and die, and the number of deaths is greater than 50%.

[0048] Table 4 shows that all plants grew normally at a salinity of 0.5%. At a salinity of 1.0%, *Potamogeton pectinata* and *Myriophyllum spicatum* were still fully viable on day 14 of the experiment. However, *Vallisneria natans*, *Elodea nuttallii*, *Hydrilla verticillata*, and *Ceratophyllum demersum* showed poor salinity tolerance, with some plants turning yellow and dying by day 7 of the experiment.

[0049] At a salinity of 2.0%, *Potamogeton pectinata* exhibited good salt tolerance, with all plants surviving by day 7 of the experiment. This was followed by *Myriophyllum spicatum*, *Vallisneria natans*, *Elodea nuttallii*, *Hydrilla verticillata*, and *Ceratophyllum demersum*, all with survival rates exceeding 50%. By day 14, most of *Vallisneria natans*, *Elodea nuttallii*, *Hydrilla verticillata*, and *Ceratophyllum demersum* had withered and died, while *Potamogeton pectinata* and *Myriophyllum spicatum* both had survival rates exceeding 50%. Therefore, the salt tolerance of the experimental plants can be ranked as follows: *Potamogeton pectinata* (survival rate 80% at 2.0% salinity) > *Myriophyllum spicatum* (survival rate 70% at 2.0% salinity) > *Hydrilla verticillata* (survival rate 20% at 2.0% salinity) > *Ceratophyllum demersum* (survival rate 10% at 2.0% salinity) > *Elodea nuttallii* (survival rate 0% at 2.0% salinity), *Vallisneria natans* (survival rate 0% at 2.0% salinity).

[0050] The results of the above salinity stress experiments show that the growth of all six submerged plant species was inhibited to varying degrees under salinity stress. However, the tolerance of different plants to hydroponic salinity differed, possibly due to variations in the development of their root and stem systems. Therefore, further testing of the plant growth and physiological indicators under different salinity water environments is necessary.

[0051] 2.1.2 Changes in relative plant height Plant height is closely related to photosynthetic efficiency, biomass, and adaptability to aquatic environments, and is an important indicator reflecting the growth of hydroponic plants. Since there was no significant difference in initial plant height among the treatment groups at the start of the experiment (p>0.05), this invention used the final plant height on day 14 and the relative plant height (with 0% salinity as the control) to evaluate the inhibitory effect of salinity stress on the growth of six hydroponic plant species. The relative plant height of the control group (0% salinity) was set at 100%. Table 5 shows that plant height generally decreased with increasing salinity. Table 6 shows that as salinity increased from 0% to 2%, the growth of all six submerged plant species was inhibited to varying degrees. At a salinity of 0.5%, the relative plant height of *Vallisneria natans* was 69%, significantly lower than the control group by 31% (p<0.05). In contrast, the relative plant heights of *Elodea nuttallii*, *Potamogeton pectinata*, *Hydrilla verticillata*, *Ceratophyllum demersum*, and *Myriophyllum sp.* did not show a significant decrease (p>0.05), indicating that *Vallisneria natans*' salt tolerance was significantly weaker than the other five tested plants. At a salinity of 1.0%, the relative plant heights of *Potamogeton pectinata* and *Myriophyllum sp.* were 103% and 93%, respectively, with no significant difference compared to the control group (p>0.05), maintaining good growth. However, the relative plant heights of *Vallisneria natans*, *Elodea nuttallii*, *Hydrilla verticillata*, and *Ceratophyllum demersum* decreased to 52%–85%, a reduction of 15%–48% compared to the control group, indicating significant growth inhibition (p<0.05). Therefore, it can be inferred that *Potamogeton pectinata* and *Myriophyllum sp.* exhibited better salt tolerance at 1.0% salinity than the other four plants. Under a salinity treatment of 2.0%, *Potamogeton pectinatus* and *Myriophyllum spicatum* showed significantly stronger salt tolerance than the other four plants, with relative plant heights of 93% and 73%, respectively (p<0.05), indicating that the growth of these two plants was not significantly inhibited. *Hydrilla verticillata*, *Ceratophyllum demersum*, and *Vallisneria natans* showed relatively strong growth inhibition and weak salt tolerance, with relative plant heights of 66%, 56%, and 53%, respectively. *Elodea nuttallii* showed the most significant growth inhibition at 45%, making it the least salt-tolerant of the six tested plants. Based on the overall growth performance of each plant under different salinity treatments, the salt tolerance of the six submerged plants was ranked as follows: *Potamogeton pectinatus* > *Myriophyllum spicatum* > *Hydrilla verticillata*, *Ceratophyllum demersum*, *Vallisneria natans* > *Elodea nuttallii*.

[0052] Table 5. Final plant height (cm) of six submerged plants under different salinity treatments.

[0053] Table 6. Relative plant height (cm) of six submerged plants under different salinity treatments.

[0054] Note: Different capital letters indicate that the relative plant height of the same plant under different salinity treatments is significantly different from that of the 0% salinity control group (p<0.05); different lowercase letters indicate that the relative plant height of different plants under the same salinity treatment is significantly different (p<0.05).

[0055] 2.1.3 Changes in relative water content of leaves The relative water content of plant leaves represents the ratio of the water content within the leaf to its maximum water capacity, reflecting the plant's ability to regulate water and its adaptability to salt stress. Table 7 shows that the relative water content of the leaves of the six submerged plants generally decreased with increasing salinity. In a saline environment, the relative water content of the leaves of all plants increased. At 0.5% salinity, *Potamogeton pectinata* showed a significant low-salt stimulation effect, with the highest relative water content of 91.18% on day 14. *Myriophyllum spicatum*, *Hydrilla verticillata*, and *Ceratophyllum demersum* maintained stable relative water content of 80%–90% at this salinity, without significant decrease (p>0.05). However, *Vallisneria natans* and *Elodea nuttallii* began to decrease to 73.21% and 72.55% respectively on day 7. The differences in salt tolerance among the six submerged plants were initially apparent under 0.5% salinity stress. At a salinity of 1.0%, *Potamogeton pectinata* and *Myriophyllum spicatum* maintained strong water retention capacity, with water contents of 72.00% and 76.42% respectively on day 14. *Hydrilla verticillata*, *Ceratophyllum demersum*, and *Vallisneria natans* saw their water contents decrease to 66.17%, 64.43%, and 62.72%, respectively, while *Elodea nuttallii* decreased to 59.30%, indicating significantly impaired water metabolism. At a salinity of 2.0%, the relative water content of *Potamogeton pectinata* and *Myriophyllum spicatum* leaves remained above 60% on day 14, at 63.53% and 62.00% respectively, demonstrating excellent osmotic regulation capacity. *Hydrilla verticillata*, *Ceratophyllum demersum*, *Vallisneria natans*, and *Elodea nuttallii* had severely lost water, with relative leaf water contents of 53.82%, 51.65%, 45.32%, and 42.94%, respectively. Therefore, the salt tolerance ranking is: Potamogeton pectinatus, Myriophyllum sp. > Hydrilla verticillata, Ceratophyllum demersum > Elodea nuttallii, Vallisneria natans, which is consistent with the previous survival rate and plant height data. 1.0% salinity is the upper limit of tolerance for most sensitive species. At 2.0% salinity, only Potamogeton pectinatus and Myriophyllum sp. have ecological restoration potential, while Vallisneria natans and Elodea nuttallii are extremely sensitive to increased salinity and are not suitable for water bodies with salinity exceeding 1.0%.

[0056] Table 7. Relative water content (%) of plant leaves under different salinity treatments

[0057] 2.2 Physiological responses of submerged plants under salt stress 2.2.1 Soluble protein content The soluble protein content of plant leaves refers to the total amount of protein dissolved in the cell sap or organelles in the leaf tissue. It is usually expressed as the mass of protein per unit fresh weight (mg / g FW) and is closely related to the plant's growth rate, nutritional status, and stress resistance. Figure 2 As shown in (a), under salt-free (control) conditions, *Hydrilla verticillata* leaves had the highest soluble protein content (4.53 mg / g FW), followed by *Elodea nuttallii* (2.07 mg / g FW), *Vallisneria natans* (1.52 mg / g FW), and *Myriophyllum spicatum* (1.37 mg / g FW). This difference is mainly attributed to the differences in genetic characteristics among species and their growth stages. Figure 2 As shown in (b)-(e), the soluble protein content of *Potamogeton pectinata* leaves was significantly higher than the control in the salinity range of 0.5%–1.0% (p<0.05). When the salinity increased to 2.0%, the soluble protein content decreased to 1.46 mg / g FW, but was still 23.73% higher than the control group. This indicates that *Potamogeton pectinata* can actively upregulate protein synthesis to cope with osmotic stress. The soluble protein content of *Vallisneria natans* and *Elodea nuttallii* both decreased significantly at salinities of 1.0% and 2.0% (p<0.05). At 2.0% salinity, the soluble protein content of *Vallisneria natans* and *Elodea nuttallii* decreased to 0.32 mg / g FW and 0.78 mg / g FW, respectively, which were 78.95% and 62.32% lower than the control group. Elodea was the most sensitive to salt stress, with a highly significant decrease in soluble protein content at all salinity treatments from 0.5% to 2.0% (p<0.01). At 2.0% salinity, the content dropped to 1.87 mg / g FW, a decrease of 58.72% compared to the control group. Ceratophyllum demersum showed no significant change in soluble protein content at 0.5% and 1.0% salinity (p>0.05), but a significant decrease at 2.0% salinity, 45.28% lower than the control group. This may be related to its needle-like leaf structure delaying salt accumulation in the tissue. Myriophyllum spicatum showed no significant difference in protein content between the control group and the treatment at any salinity (p>0.05), indicating a strong osmotic regulation capacity, which is the physiological basis for its salt tolerance.

[0058] 2.2.2 Relative conductivity The relative electrical conductivity of plant leaves is an important indicator for assessing cell membrane integrity. When plants are under stress, cell membrane structure is damaged, permeability increases, leading to an increase in relative electrical conductivity. Figure 3As shown in (a)-(d), the relative electrical conductivity of *Potamogeton pectinatus* and *Myriophyllum spicatum* was generally lower than that of other plants in the salinity range of 0.5%–2.0%. Specifically, at 0.5% salinity, *Potamogeton pectinatus* had the lowest relative electrical conductivity (9.21%); at 1.0% salinity, *Myriophyllum spicatum* had the lowest relative electrical conductivity (28.50%); and at 2.0% salinity, only *Potamogeton pectinatus* and *Myriophyllum spicatum* had relative electrical conductivity below 50%, at 42.19% and 44.40%, respectively. This indicates that *Potamogeton pectinatus* and *Myriophyllum spicatum* have higher cell membrane stability and can more effectively resist salt stress, thus their salt tolerance is better than the other four plants. In contrast, the relative electrical conductivity of *Vallisneria natans* leaves was the highest across all salinity treatments, at 20.07%, 48.54%, and 85.36% at 0.5%, 1.0%, and 2.0% salinity, respectively, indicating the most severe cell membrane damage and the worst salt tolerance. Figure 3 As shown in (e), as the salinity increased from 0% to 2.0%, the relative electrical conductivity of the leaves of the six submerged plants showed a continuous upward trend, and the differences between the salinity treatments were significant (p<0.05), indicating that salt stress caused varying degrees of damage to cell membrane integrity.

[0059] 2.2.3 Total chlorophyll content The total chlorophyll content of plant leaves is the sum of chlorophyll a and chlorophyll b. It exists on the thylakoid membrane of plant chloroplasts and is the core substance for plants to absorb light energy and participate in photosynthesis. Figure 4 The cumulative characteristics of total chlorophyll content in the leaves of six tested plant species at four different salinity levels (0%, 0.5%, 1%, and 2%) are presented. Figure 4 As shown in (a), there are significant differences in the total chlorophyll content among different plants. Under the control condition (0% salinity) and under each salinity treatment, the total chlorophyll content of Myriophyllum spicatum is the highest among all tested plants, indicating that it has a stronger photosynthetic capacity.

[0060] Depend on Figure 4As shown in (b)-(e), at a salinity of 0.5%, the total chlorophyll content of *Vallisneria natans*, *Elodea nuttallii*, and *Potamogeton pectinatus* reached its highest values, at 6.25, 8.33, and 22.95 mg / L, respectively. This indicates that slight salinity can promote photosynthesis in the leaves of some plants. At salinities of 1.0% and 2.0%, the total chlorophyll content of all tested plants showed a decreasing trend, with *Hydrilla verticillata*, *Ceratophyllum demersum*, and *Myriophyllum spicatum* showing a highly significant decrease (p<0.01). This indicates that high salinity significantly inhibits photosynthesis in these plants, with varying degrees of inhibition. Notably, under 2.0% high salinity stress, the total chlorophyll content of *Myriophyllum spicatum* was 15.80 mg / L, significantly higher than that of *Potamogeton pectinatus* and other plants. This suggests that *Myriophyllum spicatum* can mitigate the damage to the photosynthetic system caused by salt stress by regulating metabolic pathways related to chlorophyll synthesis, thereby maintaining a certain level of photosynthetic activity in high-salt environments. Therefore, *Myriophyllum spicatum* has the potential to be used as a salt-tolerant submerged plant in the ecological restoration of nearshore saline bodies.

[0061] 2.3 Comparison of nitrogen and phosphorus removal efficiency of salt-tolerant plants under different salinity levels Based on the above screening of the salt tolerance threshold of the tested submerged plants and their growth and physiological response characteristics in different salinity environments, this invention selects *Myriophyllum spicatum* and *Potamogeton crispus*, which have strong salt tolerance, as research objects to further explore their water purification capabilities in saline bodies.

[0062] 2.3.1 Effect of salinity on ammonia nitrogen removal by plants This invention investigated the effect of salinity on the ammonia nitrogen removal capacity of *Myriophyllum spicatum* and *Potamogeton crispus* by monitoring the dynamic changes in ammonia nitrogen concentration in water under different salinity treatments. Figure 5As shown in (a) and (b), the ammonia nitrogen absorption process of *Myriophyllum spicatum* and *Potamogeton pectinatus* under various salinity conditions can be divided into three stages. The first stage (0-4d) is a rapid absorption period, during which the ammonia nitrogen concentration decreases rapidly. This may be because the plants were in a relatively nitrogen-deficient state at the beginning of the experiment and needed to rapidly absorb nutrients to meet their growth and metabolic needs. The second stage (4-10d) sees a slower absorption rate, indicating that the plants have entered a period of physiological adaptation to salt stress. During this stage, they cope with environmental pressure through osmotic regulation and metabolic adjustments. The third stage (10-14d) is a dynamic equilibrium period, where the ammonia nitrogen concentration in the water bodies where the two plants are located tends to stabilize, and the absorption and release processes are basically balanced. From different salinity treatments, the ammonia nitrogen removal process of the two plants at 0.5% salinity is relatively similar to the control group. However, the ammonia nitrogen concentration of *Potamogeton pectinatus* is slightly lower than that of *Myriophyllum spicatum* in the later stages of cultivation (8-14d), showing a certain degree of low-salt adaptation. Under salinity treatments of 1% and 2%, the ammonia nitrogen removal capacity of both plants was significantly inhibited, and the removal rate was significantly slowed down, with the inhibitory effect being more pronounced at 2% salinity. Among them, the ammonia nitrogen concentration of *Myriophyllum spicatum* was consistently lower than that of *Potamogeton pectinata* in both 1% and 2% salinity water, demonstrating stronger salt stress tolerance.

[0063] Since the ammonia nitrogen concentration in each treatment group tended to stabilize in the later stage of cultivation, this invention used the removal rate index on day 14 to evaluate the ammonia nitrogen removal capacity of the two plants. The results are as follows: Figure 5 As shown in (c), at 0% salinity, the ammonia nitrogen removal rate of *Myriophyllum spicatum* was 78.10%, significantly higher than that of *Potamogeton pectinata* (62.50%), indicating a stronger nitrogen absorption capacity. At 0.5% salinity, the removal rate of *Myriophyllum spicatum* decreased to 68.49%, while that of *Potamogeton pectinata* increased to 72.90%, exhibiting a low-salt stimulation effect, which may be related to the promotion of metabolic activity in *Potamogeton pectinata* by mild salt stress. At 1% and 2% salinity treatments, the ammonia nitrogen removal rates of both plants decreased significantly with increasing salinity, but the removal rate of *Myriophyllum spicatum* was higher than that of *Potamogeton pectinata* at all salinity levels. Specifically, at 1% and 2% salinity, the removal rates of *Myriophyllum spicatum* were 51.90% and 32.80%, respectively, higher than those of *Potamogeton pectinata* (41.00% and 18.40%). This indicates that under medium-to-high salinity conditions, *Myriophyllum spicatum* can still maintain a relatively high ammonia nitrogen removal capacity, demonstrating stronger salt tolerance stability than *Potamogeton crispus*.

[0064] In summary, *Myriophyllum spicatum* not only exhibits strong salt tolerance and growth, but also maintains a relatively stable ammonia nitrogen removal capacity under salt stress, with removal rates significantly superior to *Potamogeton crispus* at both 1% and 2% salinity. This functional advantage makes *Myriophyllum spicatum* a promising candidate for ecological restoration of saline bodies.

[0065] 2.3.2 Effect of salinity on total phosphorus removal by plants By monitoring the dynamic changes in total phosphorus concentration in water bodies under different salinity treatments, this invention further investigated the effects of salt stress on the phosphorus removal capacity of *Myriophyllum spicatum* and *Potamogeton crispus*. Figure 6 As shown in (a) and (b), during the cultivation period, the removal process of total phosphorus by the two plants was similar to that of ammonia nitrogen, both experiencing a rapid absorption period (0-4d), an adaptation period (4-10d), and a balance period (10-14d). At 0% and 0.5% salinity, the total phosphorus concentration decreased rapidly in the initial stage and then leveled off, indicating that the plants gradually adapted to the environment after rapidly absorbing phosphorus to meet their growth and metabolic needs. However, at 1% and 2% salinity, the rate of decrease in total phosphorus concentration slowed significantly, and fluctuations occurred in the later stage, which may be related to tissue damage and phosphorus re-release caused by salt stress.

[0066] To further quantify the effect of salinity on the phosphorus removal capacity of the two plants, this invention calculated the total phosphorus removal rate of each treatment group on day 14. At 0% salinity, the total phosphorus removal rate of *Myriophyllum spicatum* was 66.00%, significantly higher than that of *Potamogeton pectinata* (37.86%). This result is consistent with the observations in ammonia nitrogen removal, confirming that *Myriophyllum spicatum* has superior nutrient absorption performance in a salt-free environment. Under a low-salt treatment of 0.5%, the total phosphorus removal rate of *Myriophyllum spicatum* decreased to 58.29%, while that of *Potamogeton pectinata* increased slightly to 43.25%, indicating that mild salt stress may temporarily stimulate the physiological metabolism of *Potamogeton pectinata*, but this effect did not persist at higher salinities. Under medium-high salinity treatments of 1% and 2%, the total phosphorus removal rate of both plants decreased significantly with increasing salinity. The removal rates of *Myriophyllum spicatum* at 1% and 2% salinity were 43.80% and 36.09%, respectively, while those of *Potamogeton crispus* decreased to 32.75% and 25.40%, respectively. This indicates that high salt stress significantly weakens the plant's ability to absorb nutrients by disrupting cell membrane integrity and inhibiting photosynthesis.

[0067] Notably, under all salinity gradients, *Myriophyllum spicatum* exhibited a higher total phosphorus removal rate than *Potamogeton crispus*, demonstrating stronger salt stress tolerance and functional stability. *Myriophyllum spicatum* not only excels in ammonia nitrogen removal but also shows a significant advantage in total phosphorus removal. This synergistic removal capability of multiple nutrients makes it highly valuable for the ecological restoration of eutrophic saline bodies.

[0068] This invention systematically studies the salt tolerance and water purification capabilities of six common submerged plants (Vallisneria natans, Elodea nuttallii, Potamogeton pectinatus, Hydrilla verticillata, Ceratophyllum demersum, and Myriophyllum sp.) found in estuaries and nearshore waters around Guangdong. First, the survival, growth, and physiological responses of the six submerged plants under different salinity gradients (0%, 0.5%, 1%, and 2%) were evaluated. Then, Myriophyllum sp. and Potamogeton pectinatus, which exhibit strong salt tolerance, were selected as research subjects, and their ammonia nitrogen and total phosphorus removal capabilities under different salinity stresses during a 14-day cultivation period were compared. The main conclusions are as follows: A salinity of 1.0% is the key threshold for distinguishing between salt-tolerant and sensitive species. Within the salinity range of 0-0.5%, all six plant species can grow normally; when the salinity rises to 1.0%, only *Myriophyllum spicatum* and *Potamogeton crispus* survive completely, while the other four species show partial yellowing and death; at a salinity of 2.0%, only these two plants maintain partial survival (survival rate >50%), while most of the other species die.

[0069] As salinity increased, plant growth was generally inhibited, and significant differences in salt tolerance were observed among species. At 0.5% salinity, the growth of the other five plant species, except for *Vallisneria natans*, was not significantly affected, while *Potamogeton pectinata* exhibited a low-salt stimulation effect. At 1.0% and 2% salinity, only *Potamogeton pectinata* and *Myriophyllum sp.* maintained good growth and water balance, while the growth of the other species was inhibited and water metabolism was impaired. The salt tolerance of the six submerged plants was ranked as follows: *Potamogeton pectinata* > *Myriophyllum sp.* > *Hydrilla verticillata*, *Ceratophyllum demersum* > *Vallisneria natans*, *Elodea nuttallii*. Only *Potamogeton pectinata* and *Myriophyllum sp.* possessed potential for ecological restoration in high-salt environments.

[0070] The differences in physiological indicators further validated the differentiation in salt tolerance among species. *Potamogeton pectinata* actively upregulated soluble protein synthesis to cope with osmotic stress under salt stress; *Myriophyllum spicatum* maintained stable protein content across all salinity levels, with a relative conductivity consistently below 50%, and exhibited the highest total chlorophyll content among all treatment groups, demonstrating stable cell membrane structure and strong photosynthetic capacity. *Vallisneria natans* and *Elodea nuttallii* showed significant deterioration in physiological indicators under high salt conditions, exhibiting the weakest salt tolerance.

[0071] In terms of water purification capacity, the nitrogen and phosphorus removal efficiency of *Myriophyllum spicatum* is significantly better than that of *Potamogeton pectinata*. The removal rates of ammonia nitrogen and total phosphorus for both showed a slowing trend with the extension of treatment time, and the removal efficiency eventually stabilized. Under various salinity gradients, *Myriophyllum spicatum* had higher removal rates of ammonia nitrogen and total phosphorus than *Potamogeton pectinata*, and maintained high purification efficiency even in high salinity environments.

[0072] Based on the comprehensive analysis of salt tolerance and water purification capacity, *Potamogeton pectinata* exhibits the strongest salt tolerance, but its nitrogen and phosphorus removal capacity is weaker than that of *Myriophyllum spicatum*. *Myriophyllum spicatum* combines strong salt tolerance with highly efficient nitrogen and phosphorus removal capabilities, making it more suitable for ecological restoration in nearshore brackish water areas. Therefore, this invention identifies *Myriophyllum spicatum* as the appropriate experimental material.

[0073] 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 rapid screening and comprehensive evaluation of salt-tolerant submersed macrophytes in estuary, characterized in that, Includes the following steps: Step 1: The submerged plants to be screened were cultured under artificially simulated salt stress conditions with different salinity gradients, and the survival rate, growth indicators and physiological indicators of the plants were recorded. Step 2: Determine the salt tolerance survival threshold for each plant based on its survival rate and growth indicators; Step 3: Analyze the physiological response characteristics of each plant under salt stress based on physiological indicators; Step 4: Select plants with strong salt tolerance and further determine their removal efficiency of nitrogen and phosphorus nutrients in water. Step 5: Based on the combination of salt tolerance and nutrient removal capacity, the submerged plants with the best overall performance are selected.

2. The method of claim 1, wherein, In step 1, the salinity gradient is 0%, 0.5%, 1.0%, and 2.0%; the cultivation conditions are set as follows: temperature 25±2℃, light intensity 4000 lux, light-dark cycle 12h / 12h, and cultivation period 14 days; the submerged plants include at least one of Vallisneria natans, Elodea nuttallii, Potamogeton pectinatus, Hydrilla verticillata, Ceratophyllum demersum, and Myriophyllum spicatum.

3. The method of claim 1, wherein, In step 1, the survival rate is determined by the following criteria: complete loss of chlorosis of the plant leaves and softening and lodging of the stems are used as the basis for determining death; the growth indicators include plant height and relative water content of leaves; the physiological indicators include soluble protein content, relative conductivity and total chlorophyll content of leaves.

4. The method of claim 1, wherein, In step 2, the criterion for determining the salt tolerance survival threshold is: the survival rate of the plant after 14 days of cultivation at a certain salinity is ≥50% as the basis for its ability to tolerate that salinity.

5. The method of claim 1, wherein, In step 3, the analysis of the physiological response characteristics includes: comparing the changes in the soluble protein content of leaves of various plants under different salinity treatments, the degree of damage to cell membrane integrity, and the total chlorophyll content; the cell membrane integrity is characterized by relative conductivity.

6. The method of claim 1, wherein, In step 4, the method for determining the nitrogen and phosphorus nutrient removal efficiency is as follows: in a static simulation experiment, monitor the dynamic changes of ammonia nitrogen and total phosphorus concentrations in the water under different salinity conditions, and calculate the ammonia nitrogen removal rate and total phosphorus removal rate within a 14-day culture period.

7. The method of claim 2, wherein, The submerged plant with the best overall performance mentioned in step 5 is Myriophyllum spicatum.

8. A method of screening salt-tolerant submerged plants suitable for ecological restoration of coastal wetlands, estuarine areas or brackish water areas, characterized in that, The rapid screening and comprehensive evaluation method for salt-tolerant submerged plants in estuaries as described in any one of claims 1-7 is adopted.

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