Method for enhancing phytoremediation capability of salix matsudana on Cd pollution by exogenously adding 5-aminolevulinic acid and application

Abstract

The invention discloses a method for enhancing the phytoremediation capacity of salix matsudana on Cd pollution by exogenously adding 5-aminolevulinic acid (5-ALA) and application. According to the method, the 30 mg / L 5-ALA aqueous solution is sprayed to the leaf surfaces of the salix matsudana in a water mist mode, so that plant growth is promoted, the Cd resistance of the plants is improved, the absorption, accumulation and transportation capacities of the plants to heavy metal Cd are enhanced, and the phytoremediation efficiency is improved. The method provided by the invention has wide practicability, and provides a new thought and method for the practice of repairing the Cd-polluted soil by utilizing a phytoremediation technology.

Description

[0001] The invention is supported by the National Natural Science Foundation of China (31901184), Tianjin Natural Science Foundation (17JCYBJC22500) and Tianjin Key Experiment Open Fund for Animal and Plant Resistance. Technical Field

[0002] The invention belongs to the field of biotechnology, and in particular relates to a method for enhancing the phytoremediation ability of plants to Cd-contaminated soil and an application thereof. Background Art

[0003] Heavy metal cadmium (Cd) pollution has become a global concern. Cd pollution seriously threatens the ecological environment and human health. Cd is a non-essential element for plant growth. It is absorbed into the plant body through the plant roots and will accumulate in animals and even humans through the food chain, thus affecting human health. Therefore, it is urgent to control Cd pollution. The methods for controlling heavy metal pollution are divided into physical remediation, chemical remediation and biological remediation (including phytoremediation, microbial remediation, etc.).

[0004] Phytoremediation refers to the biological purification of the environment (soil, water and air) by plants. Phytoremediation is generally considered to be a green technology because of its good heavy metal purification ability and minimal secondary waste into the environment. In addition, phytoremediation is highly accepted by the public due to its easy application, low cost and environmental friendliness, and has become a hot area of ​​research and development at home and abroad. So far, more than 450 heavy metal hyperaccumulators have been discovered. However, most of the hyperaccumulators discovered are herbaceous, with small aboveground biomass, small amount of heavy metals accumulated and transferred, long repair time and low efficiency, which directly limits the effect of phytoremediation. Therefore, heavy metal hyperaccumulators are generally only used as materials for the study of heavy metal absorption and tolerance mechanisms. Woody plants are not as good as herbaceous hyperaccumulators in terms of their ability to accumulate heavy metals. However, perennial, tall and fast-growing woody plants such as poplars and willows have more obvious advantages in the treatment of heavy metal-contaminated soils due to their large biomass, well-developed root systems and non-connection to the food chain, making them more suitable as candidate plants for phytoremediation.

[0005] Due to the current low efficiency of phytoremediation, phytoremediation technology combined with other technologies for joint restoration has become the focus of current research, such as the application of exogenous substances to improve plant resistance to Cd. Exogenous substances assisted phytoremediation can enhance the absorption and accumulation of heavy metal Cd by restoration plants to a certain extent, such as chelating agents, surfactants, biochar, salicylic acid, nitric oxide (NO), polyamines, etc., by regulating the distribution and accumulation of heavy metals in the body, reducing the level of oxidative damage caused by heavy metals, and enhancing the tolerance of plants to heavy metals to improve the efficiency of phytoremediation.

[0006] 5-Aminolevulinic acid (5-ALA) is an organic compound containing oxygen and nitrogen. It is the common precursor of the biosynthesis of all porphyrin compounds. It is a non-protein amino acid widely found in bacteria, fungi, animals and plants. It is a naturally occurring physiologically active substance in plants that is essential for plant life activities and has active metabolism. It has no toxic side effects, is easily degraded and has no residue. It is a beneficial plant growth regulator that can enhance the ability of plants to resist abiotic stress.

[0007] By exogenously adding 5-ALA to plants under heavy metal Cd stress in a suitable manner, plant growth and plant resistance are promoted, and the plant's ability to absorb, accumulate and transport Cd is enhanced, thereby improving the efficiency of plant remediation. Therefore, the present invention will provide new ideas and technologies for using woody plants to repair soil heavy metal Cd pollution. Summary of the invention

[0008] The purpose of the present invention is to overcome the disadvantage of low efficiency of existing phytoremediation, and disclose a method and application of enhancing the phytoremediation ability of Salix matsudana to Cd pollution by spraying 5-ALA on leaves.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] Provided is a method for enhancing the phytoremediation ability of Salix matsudana for Cd pollution by foliar spraying of 5-ALA, characterized by providing a suitable exogenous 5-ALA addition mode, concentration, dosage and application time; comparing the target plant with the control plant 28 days after application, comparing the growth parameters, Cd resistance physiological indicators, Cd absorption accumulation and transport capacity of the two, and then comparing the phytoremediation efficiency of the two.

[0011] To this end, the present invention discloses the following technical contents:

[0012] A method for enhancing the phytoremediation ability of Salix matsudana to Cd pollution by exogenously adding 5-aminolevulinic acid (5-ALA), characterized by providing a suitable exogenously added 5-ALA method, concentration, dosage and application time. Method: spraying 5-ALA aqueous solution on the leaf surface of Salix matsudana in the form of water mist (since 5-ALA is easily decomposed by light, its aqueous solution is packaged in black bottles and stored away from light); concentration: 30 mg / L; spraying time: 17:00-19:00 every afternoon; each spraying dosage: 4 mL / plant, spraying until droplets are about to drip from the leaves.

[0013] The present invention further discloses the application of enhancing the phytoremediation ability of Salix matsudana to Cd-contaminated soil by exogenous addition of 5-ALA. The application refers to spraying a certain amount of 5-ALA aqueous solution on the leaf surface of Salix matsudana, which can significantly promote the growth of Salix matsudana under Cd stress, improve its absorption and accumulation ability and resistance to heavy metal Cd, and thus improve the phytoremediation efficiency of Salix matsudana to Cd-contaminated soil.

[0014] The method provided by the present invention for enhancing the phytoremediation ability of Salix matsudana to Cd pollution by exogenous addition of 5-ALA has the beneficial effect, compared with the prior art, that by spraying a certain dose of 5-ALA on the leaf surface of Salix matsudana, the growth of Salix matsudana can be significantly promoted, and its absorption, accumulation and resistance to heavy metal Cd can be improved. The present invention provides a new idea and method for the phytoremediation practice of Cd pollution.

[0015] The present invention further discloses the application of spraying 5-ALA on leaves to enhance the phytoremediation efficiency of Salix matsudana against Cd pollution and to alleviate the Cd toxicity of Salix matsudana. The experimental results show that: Figure 1 The dry weight, fresh weight and relative plant height of Salix matsudana in different treatment groups. As can be seen from the figure, compared with the control group, Cd severely inhibited the plant height of Salix matsudana, and 5-ALA spraying significantly alleviated this growth inhibition. At the same time, 5-ALA foliar spraying greatly promoted the increase of Salix matsudana biomass (fresh weight, dry weight). Figure 2 The photosynthetic pigment content and photosynthetic fluorescence parameters of Salix matsudana leaves in different treatment groups. As can be seen from the figure, Cd treatment reduced the carotenoid and chlorophyll b contents, and also reduced the values ​​of photosynthetic fluorescence parameters such as the photochemical quenching coefficient (qP), the actual quantum efficiency of the open center under light adaptation (Y(II)), and the effective photochemical quantum yield of PSII (Fv' / Fm'), and increased the value of the non-photochemical fluorescence quenching coefficient (qN). Compared with the Cd treatment group, the carotenoid content, chlorophyll b content, qP, Y(II) and Fv' / Fm' values ​​of Salix matsudana leaves increased after spraying 30 mg / L 5-ALA, and the qN value decreased. This means that Cd inhibited the photosynthesis of Salix matsudana, while 5-ALA spraying alleviated the inhibitory effect of Cd treatment on the photosynthesis of Salix matsudana leaves. Figure 3The antioxidant enzyme activities and malondialdehyde contents of Salix matsudana in different treatment groups. Compared with the control group, spraying 5-ALA significantly increased the activities of three antioxidant enzymes of Salix matsudana (P < 0.05), including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Malondialdehyde (MDA) represents the degree of membrane lipid peroxidation. Therefore, the higher the MDA content, the higher the degree of peroxidation. Compared with the control group, Cd treatment significantly increased the MDA content of Salix matsudana, while the MDA content was significantly decreased after exogenous application of 5-ALA (P < 0.05). Table 1 shows the Cd content of Salix matsudana roots, stems, and leaves after 28 days of different treatments. The Cd content of each organ was: root > stem > leaf. Comparing the Cd single treatment group with the Cd + 5-ALA group, it was found that the Cd content of the roots, stems, and leaves increased. Therefore, foliar spraying of 5-ALA promoted the growth and physiological resistance of Salix matsudana to Cd and enhanced the restoration capacity of Salix matsudana to Cd pollution, which provided theoretical support for the phytoremediation practice of Cd pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Dry weight, fresh weight and relative plant height of Salix matsudana in different treatment groups.

[0017] Figure 2 Photosynthetic pigment content and photosynthetic fluorescence parameters of Salix matsudana leaves in different treatment groups.

[0018] Figure 3 Antioxidant enzyme activities and malondialdehyde content in Salix matsudana in different treatment groups.

[0019] Table 1 shows the Cd contents in roots, stems and leaves of Salix matsudana after 28 days of different treatments.

[0020] For ordinary technicians in this field, other relevant drawings can be obtained based on the above drawings without any creative work. DETAILED DESCRIPTION

[0021] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described below in conjunction with specific embodiments.

[0022] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments:

[0023] The various raw materials and equipment used in the present invention are all conventional commercially available products and can be directly purchased from the market.

[0024] The sources of the biomaterials used in the examples are as follows: Salix matsudana was grown in the Science and Technology Park of Tianjin Normal University. 5-ALA was purchased.

[0025] Embodiment 1:

[0026] Plant cultivation

[0027] Experimental materials: Salix matsudana was taken from the Biotechnology Park of Tianjin Normal University. The branches of Salix matsudana with uniform thickness, vigorous growth and health were selected, and the length was about 20 cm, and soil cuttings were cultivated. The soil and vermiculite were mixed in a ratio of 3:1 for soil cultivation, and each pot of soil weighed about 250g. The soil without Cd pollution was used as the control group, and the soil contaminated with Cd was used as the experimental group (the final concentration of Cd was 50mg / kg). Three branches were planted in each pot, and the cutting depth was approximately 1 / 3 of the branch. There were four groups in the experiment, namely the control group, the single Cd treatment group, the group sprayed with 5-ALA only, and the Cd treatment + sprayed with 5-ALA group, named as: CK, Cd, 30ALA, Cd+30ALA. Five pots were set up for each group. Watering was done every day to keep the soil moist to promote the rooting of Salix matsudana branches and the growth of young new branches.

[0028] Embodiment 2:

[0029] Exogenous addition of 5-ALA

[0030] When all the newly sprouted branches in each group have grown to be strong and the leaves have grown to maturity, the 5-ALA aqueous solution was sprayed on the leaves of the 30ALA and Cd+30ALA groups of Salix matsudana in the form of water mist. The leaves of the control group and the single Cd treatment group were sprayed with water in the same way. After preliminary experimental screening, it was found that the appropriate concentration of 5-ALA was 30 mg / L (because 5-ALA is easily decomposed by light, its aqueous solution was packaged in black bottles and stored away from light), the best spraying time was 17:00-19:00 every afternoon, and the spraying dose was 4 mL each time, and it was sprayed until droplets were about to drip from the leaves.

[0031] Embodiment 3:

[0032] Determination of physiological indicators

[0033] (1) Chlorophyll content

[0034] After 28 days of foliar spraying, the height of each group of Salix matsudana was measured, and the fresh weight (FW) of Salix matsudana leaves was 0.2g, and the leaves were ground with 10mL 80% acetone in the dark. The mixture was then poured into a conical flask, sealed in the dark at 4℃ for 12h, and the supernatant was taken and centrifuged (4000r / min) for 20min. The extract was analyzed using a spectrophotometer (UV / Vis Spectrometer, PerkinElmerLambda 35, Boston, USA). The absorbance of the sample was read on the spectrophotometer at wavelengths of 663, 646 and 470nm, respectively.

[0035] The formulas for calculating chlorophyll and carotenoid content are as follows:

[0036] Chlorophyll a (mg / g) = (12.21 × A663 - 2.81 × A646) / FW

[0037] Chlorophyll b (mg / g) = (20.13 × A646 - 5.03 × A663) / FW

[0038] Carotenoids

[0039] (mg / g)=[1000×A470-3.27×(12.21×A663-2.81×A646)-104(20.13×A646-5.03)] / (229·FW)

[0040] (2) Determination of photosynthetic fluorescence parameters

[0041] After 28 days of foliar spraying, the photosynthetic fluorescence parameters of Salix matsudana leaves were measured using a portable fluorometer (Dual-Pam / F, Walz, Ger). The leaves were darkened for 20 minutes before measurement. The fifth fully expanded leaf from the top was selected for each measurement, and each measurement was repeated 5 times. The measurement parameters were as follows: F0, Fm, Fv / Fm, actual quantum efficiency of the open center of PSII under light adaptation (Y(II)), photochemical coefficient of quenching (qP), and non-photochemical coefficient of quenching (qN).

[0042] (3) Determination of antioxidant enzyme activity

[0043] After 28 days of foliar spraying, take 0.5g of fresh and clean leaves, wash with distilled water, dry with filter paper, put in a mortar, add 10mL of PBS solution, homogenize on ice, and place in a centrifuge tube after grinding. Centrifuge at 4℃, 13000r for 20min, and take the supernatant (enzyme solution) to test the enzyme activity. The activity of superoxide dismutase (SOD) is determined by the nitroblue tetrazolium method. The activity of peroxidase (POD) is determined by the guaiacol method. The activity of catalase (CAT) is determined by the hydrogen peroxide decomposition method. The determination of the activities of these three antioxidant enzymes are all common plant physiology methods.

[0044] (4) Determination of malondialdehyde (MDA) content

[0045] After 28 days of foliar spraying, take 0.5 g of fresh and clean leaves, wash with distilled water, dry with filter paper, add 10 mL of 10% trichloroacetic acid (TCA) solution in a mortar, centrifuge at 4000r for 15 min at 4°C, take 2 mL of the upper clear layer, replace the control group with 2 mL of distilled water, add 2 mL of 0.6% 2-thiobarbituric acid (TBA, prepared with 10% TCA) solution, mix well, boil in a boiling water bath for 15 min, and perform color reaction. After cooling, use a spectrophotometer (UV-visible spectrophotometer UV-1800PC, MAPADA, Shanghai) to record the absorbance (OD value) at 450nm and 532nm to calculate the MDA content.

[0046] MDA content (μmol / g) = [total volume of extract (6.45 × OD 532 -0.56×OD 450 )] / (volume of extract × fresh weight of sample during determination)

[0047] Embodiment 4:

[0048] Determination of Cd content

[0049] (1) Material collection: 28 days after foliar spraying, pull out the soil-grown plants with their roots and rinse the roots with running water until they are completely rinsed. Then separate the roots, stems, and leaves, wash them with distilled water, wipe them dry with filter paper, cut them into pieces, weigh them, and record their fresh weight. Then place them in a clean culture dish and move them into an oven for drying.

[0050] (2) Sample drying: Dry at 45°C for 3 days, 80°C for 2 days, and 105°C for 12 hours until the sample weight is constant. Measure the dry weight of the sample.

[0051] (3) Grinding: Grind the dried sample into powder using a grinder and store it in a sealed glass container.

[0052] (4) Sample digestion: Weigh 0.2000 g of each sample, add 10 mL of concentrated nitric acid and 2 mL of perchloric acid, and perform wet digestion in a microwave oven until the organic matter is completely decomposed and colorless or white crystals are precipitated.

[0053] (5) Make up the volume: Use 10% dilute nitric acid to accurately make up the volume of the digested sample into a 25 mL volumetric flask.

[0054] (6) ICP-OES determination of Cd content: The Cd content of each sample was determined using an ICP-OES instrument, and the Cd content of the root, stem, and leaf of each treatment group was calculated. SPSS 19.0 (SPSS Inc, Illinois, USA) was used to analyze the data. The data are expressed as mean ± standard error (SE). One-way analysis of variance (ANOVA) was used to test the significance of differences between different treatment groups. The results are shown in Table 1.

[0055] Table 1. Cd content in roots, stems and leaves of Salix matsudana after 28 days of different treatments

[0056]

[0057] The present invention is described above by way of example. It should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by those skilled in the art without inventive effort falls within the protection scope of the present invention.

Claims

1. A method for enhancing the phytoremediation capacity of Salix matsudana for Cd pollution by exogenously adding 5-aminolevulinic acid (5-ALA), characterized in that Provide appropriate exogenous 5-ALA addition methods, concentrations, dosages and application times. Method: Spray 5-ALA aqueous solution on the leaves of Salix matsudana in the form of water mist (since 5-ALA is easily decomposed by light, its aqueous solution is packaged in black bottles and stored away from light). Concentration: 30 mg / L; Spraying time: 17:00-19:00 every afternoon; Each spraying dosage: 4 mL / plant, spray until droplets are about to drip from the leaves.

2. Application of the method of exogenous addition of 5-ALA according to claim 1 in enhancing the phytoremediation of Cd-contaminated soil by Salix matsudana.

3. The application described in claim 2 refers to spraying 5-ALA on the leaves of Salix matsudana in the manner, concentration, time and dosage described in claim 1, which can significantly promote the growth of Salix matsudana under Cd stress, improve its absorption and accumulation capacity and resistance to heavy metal Cd, thereby improving the phytoremediation efficiency of Salix matsudana on Cd-contaminated soil.

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