Method for preventing and controlling transfer of cadmium in hyperaccumulator

Abstract

The invention relates to the field of soil remediation, in particular to a method for preventing and controlling transfer of cadmium in hyperaccumulators, which comprises the following steps: planting annual ryegrass in cadmium-containing soil, and applying a leaf surface preventing and controlling agent to the annual ryegrass. According to the method, the leaf surface resistance and control agent is added into the annual ryegrass, the enrichment capacity of the underground part of the plant to cadmium is enhanced through the leaf surface resistance and control agent, transfer of cadmium of the underground part of the plant to the overground part of the plant is reduced, a large amount of biomass polluted by cadmium is prevented from being generated, and the cadmium of the overground part of the plant meets the regulation of pollutant limit in food in the national food safety standard.

Description

Technical Field

[0001] The invention relates to the field of soil remediation, and in particular to a method for blocking and controlling cadmium transport in hyperaccumulator plants. Background Art

[0002] Cadmium is a toxic heavy metal that can accumulate through the food chain even at low concentrations and cause serious impacts on human health, such as kidney damage and osteoporosis. The cadmium content in soil and water in many parts of China exceeds the standard, which directly affects the safety and quality of food. According to environmental monitoring data, cadmium pollution is particularly serious in some areas in southern China, especially in some heavy industrial areas and areas rich in mineral resources. Therefore, the remediation of soil Cd pollution is of great significance to food security and human health.

[0003] Currently, phytoremediation has attracted increasing interest in eliminating heavy metals present in soil due to its advantages in field application and eco-friendliness. Phytoextraction, phytostabilization, phytofiltration, phytovolatilization, photodegradation and phytodegradation all belong to phytoremediation. Among them, plant extraction is to plant hyperaccumulators in contaminated soil, absorb heavy metals in the soil into the body through the enrichment ability of plants, and reduce the content of heavy metals in the soil. However, after plant extraction and remediation, heavy metals will be enriched in the aboveground part of the plant in large quantities. It is also difficult to treat the aboveground biomass enriched with heavy metals in a green and pollution-free way. Moreover, the soil used to plant hyperaccumulators cannot generate economic benefits for a long time. Plant stabilization relies on special substances secreted by the plants themselves to stabilize heavy metals and prevent them from further migration and diffusion into the food chain. However, heavy metals always remain in the soil and cannot be completely removed. Moreover, with the changes in environmental factors, they may become active again and be absorbed by plants. Plant volatilization is to convert heavy metals into volatile forms and release them into the atmosphere to remove pollution sources from the soil. However, the research on plant volatilization remediation is still in its early stages. Some volatilized substances of plants may also produce other toxins that act on the atmosphere, thereby polluting the atmosphere. Annual ryegrass is one of the plants with high cadmium accumulation in roots. Due to the high level of cadmium accumulation in the roots, it is a good plant remediation material.

[0004] Therefore, there is an urgent need for a soil remediation method that can reduce the cadmium concentration in the soil through plant enrichment without producing a large amount of difficult-to-handle cadmium-enriched biomass, and can improve soil utilization and prevent the soil from having no economic benefits for a long time. Summary of the invention

[0005] In view of the technical problems in the above-mentioned prior art, a method for preventing and controlling cadmium transport in hyperaccumulator plants is provided. By adding foliar inhibitors to annual ryegrass, the foliar inhibitors are used to enhance the cadmium enrichment capacity of the underground part of the plant, reduce the transfer of cadmium from the underground part of the plant to the aboveground part of the plant, prevent the production of a large amount of cadmium-contaminated biomass, and make the cadmium in the aboveground part of the plant meet the requirements of the National Food Safety Standard Limits of Contaminants in Food (GB2762-2017).

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A method for controlling cadmium transport in hyperaccumulator plants comprises planting annual ryegrass in soil containing cadmium and applying a foliar control agent to the annual ryegrass.

[0008] Annual ryegrass is a plant that hyperaccumulates cadmium. It can enrich cadmium in the soil outside its body, thereby reducing the cadmium content in the soil. By adding foliar barrier to annual ryegrass, the cadmium enriched in annual ryegrass is fixed in the underground root system, reducing the amount of cadmium transferred to the aboveground part of the plant, greatly reducing the amount of cadmium-enriched biomass, and reducing the difficulty and cost of later treatment; and the cadmium content in the upper part of annual ryegrass is lower than the provisions of the "National Food Safety Standard Limits of Contaminants in Food" (GB2762-2017), enhancing the safety of the upper part of annual ryegrass.

[0009] Preferably, the foliar barrier agent is any one or more of sodium selenite, NaHS hydrate, sodium nitroprusside, S-allyl-L-cysteine, sodium silicate and salicylic acid.

[0010] Preferably, the foliar barrier agent is sodium nitroprusside.

[0011] Preferably, the foliar barrier agent is applied by any one or more of foliar spraying or root injection.

[0012] Preferably, a microbial composite agent is also added to the roots.

[0013] Preferably, the microbial composite agent comprises: 15-25% of Bacillus subtilis, 5-15% of Bacillus cereus and 15-25% of Citrobacter rodi.

[0014] Preferably, the microbial composite agent comprises: 20% Bacillus subtilis, 10% Bacillus cereus and 20% Citrobacter rodi.

[0015] Preferably, the cadmium content in the soil is 0.1-10 mg / L -1 .

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention adds a foliar barrier to annual ryegrass, and uses the foliar barrier to enhance the cadmium enrichment capacity of the plant's underground part, prevent the cadmium enriched in the plant's underground part from transferring to the plant's aboveground part, and prevent the generation of a large amount of cadmium-contaminated biomass, so that the cadmium in the plant's aboveground part meets the requirements of the National Food Safety Standard Limits of Contaminants in Food (GB2762-2017). BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Diagram of sowing seeds for plants.

[0019] Figure 2 The figure shows the effects of six foliar barrier agents on the dry weight of annual ryegrass.

[0020] Figure 3 Graph showing the effects of six foliar inhibitors on cadmium uptake by annual ryegrass.

[0021] Figure 4 Figure 2 shows the effects of six foliar inhibitors on cadmium transport in annual ryegrass.

[0022] Figure 5 The effect of spraying foliar inhibitors NO, Se, and Si on four metal cations in annual ryegrass (a: cadmium concentration of 3 mg kg -1 Changes of Ca, Mg, Zn, and Cu in the upper part of annual ryegrass in soil; b: Cadmium concentration of 3 mg kg -1 Changes of Ca, Mg, Zn, and Cu in the lower part of annual ryegrass in the soil; c: Cadmium concentration of 5 mg kg -1 Changes of Ca, Mg, Zn, and Cu in the upper part of annual ryegrass in soil; d: Cadmium concentration of 5 mg kg -1 Changes of Ca, Mg, Zn, and Cu in the lower part of annual ryegrass in the soil; e: Cadmium concentration is 10 mg kg -1 Changes of Ca, Mg, Zn, and Cu in the upper part of annual ryegrass in soil; f: Cadmium concentration is 10 mg kg -1 Changes of Ca, Mg, Zn and Cu elements in the lower part of annual ryegrass in soil).

[0023] Figure 6 This is a correlation analysis chart of the contents of Cd and four divalent cations after spraying foliar control agents NO, Se and Si.

[0024] Figure 7 The effect of foliar inhibitor NO on dry weight and cadmium content of annual ryegrass (a: cadmium concentration is 3 mg kg -1Dry weight of the upper and underground parts of annual ryegrass in soil; b: Cadmium concentration of 5 mg kg -1 Dry weight of the upper and underground parts of annual ryegrass in soil; c: cadmium concentration of 10 mg kg -1 Dry weight of the upper and lower parts of annual ryegrass in soil; d: cadmium concentration of 3 mg kg -1 Changes in cadmium content in the upper and underground parts of annual ryegrass in the soil; e: cadmium concentration of 5 mg kg -1 Changes in cadmium content in the upper and underground parts of annual ryegrass in the soil; f: cadmium concentration of 10 mg kg -1 Changes in cadmium content in the aboveground and underground parts of annual ryegrass in soil).

[0025] Figure 8 Figure 2. Changes in cadmium accumulation in the roots of annual ryegrass with different additions of foliar inhibitor NO (a: cadmium concentration of 3 mg kg -1 Figure 2. Cadmium accumulation in the lower part of annual ryegrass in soil; e: Cadmium concentration of 5 mg kg -1 Figure 2. Cadmium accumulation in the lower part of annual ryegrass in soil; f: Cadmium concentration of 10 mg kg -1 Figure 2. Cadmium accumulation in the lower part of annual ryegrass in soil). DETAILED DESCRIPTION

[0026] In order to more clearly describe the invention purpose, technical scheme and technical effect advantages in the specific implementation case of the present invention, the scheme in the specific embodiment will be described in detail in combination with the drawings of the specification of the present invention. The specific technical scheme involved in the following specific embodiments is only for a clear and complete description of the innovative technical scheme of the present invention. It is only a part of the specific implementation scheme that can be adopted by the present invention, not all embodiments, and should not be understood as a limitation on the innovative scheme of the present invention. Any scheme adopting the same inventive concept of the present invention should be included in the protection scope of the present invention.

[0027] Secondly, the relevant descriptions of the drawings in the specific embodiments of the present invention are only for the convenience of technical personnel to understand the scheme of the present invention. Some details in the drawings are displayed for the convenience of clearly presenting the technical scheme. It should not be considered that all technical features in the drawings must be included in the specific implementation cases, and the detailed features in the drawings cannot be regarded as additional limitations on the innovative technical scheme of the present invention.

[0028] For those skilled in the art, when understanding the solutions described in the specific embodiments of the present invention, they can refer to the conventional technical manuals in the field. At the same time, for the places where the above-mentioned terms appear, they can make appropriate understandings or adjustments for reference, and deduce the implementation of the same or similar technical solutions without paying any creative work.

[0029] Example 1

[0030] 1 Materials and methods

[0031] 1.1 Experimental Materials

[0032] Annual ryegrass (Lolium multiflorum Lam.) from Jiangsu Xintiandi Seed Co., Ltd. was selected.

[0033] Types of foliar inhibitors: Sodium selenite (Se), NaHS hydrate (S), sodium nitroprusside (NO donor), and S-allyl-L-cysteine ​​(SAC) were purchased from Aladdin Reagent (Shanghai) Co., Ltd.; sodium silicate (Si) and salicylic acid (SA) were purchased from Chengdu Cologne Chemical Company.

[0034] The soil used in the experiment was taken from Mianyang City, Sichuan Province. The background content of cadmium (Cd) in the soil was 0.18 mg kg -1 , pH value is 7.29. Its moisture content is 12.8%, and the organic matter content reaches 22.30 mg kg -1 , alkaline nitrogen is 53.74 mg kg -1 The available phosphorus content and available potassium content were 7.31 mg kg -1 and 21.36 mg kg -1 .

[0035] 1.2 Preparation of Cd-contaminated soil

[0036] Select Cd(NO 3 ) 2 , set the Cd concentration to 10 mg kg -1 , with no Cd added as the control group. Cd solutions of different concentrations were prepared according to the Cd gradient setting, and the Cd solutions were evenly sprayed into the soil and then placed in a dark place for 30 days to ensure that the soil reached a balanced state, thus completing the preparation of Cd-contaminated soil.

[0037] 1.3 Potted plant experiment

[0038] Set 10mg·kg -1 and blank control (CK, 10 mg kg -1 The potted plant test container was a round flower pot with a hole and a tray at the bottom, and each pot was filled with 1 kg of soil according to the water content.

[0039] like Figure 1As shown, seeds with full grains were selected and soaked in deionized water overnight, then placed in a flowerpot, covered with a thin layer of soil and controlled with appropriate temperature and humidity (water holding capacity was maintained at 65% to 75%) during this period. The plants were harvested when they just headed, and relevant indicators were measured.

[0040] 1.4 Experimental design

[0041] Sowing: Disinfect and soak the annual ryegrass seeds, then wash them with deionized water. Sow 55 seeds in each pot and observe their growth. After germination, keep 50 seedlings of the same size in each pot during the planting period.

[0042] Foliar barrier screening: In this experiment, six cadmium transport barrier agents were selected, sodium selenite (Se), NaHS hydrate (S), sodium nitroprusside (NO donor), S-allyl-L-cysteine ​​(SAC), sodium silicate, and salicylic acid (SA), with three concentration gradients each, and three replicates for each experiment, see Table 1. The exogenous foliar spray barrier agent was sprayed on the leaf surface of annual ryegrass in the form of an aqueous solution, and the upper and lower surfaces of the leaves were evenly misted, and the liquid did not flow down. After the plant grew for 15 days, the solution was evenly sprayed on the leaves according to the corresponding concentration, and the frequency of spraying the foliar barrier agent was every 7 days. This study selected natural soil without cadmium (CK) and soil contaminated with cadmium (CKK). During the experiment, deionized water was used instead of foliar barrier agents for spraying. A total of 19 groups of experiments were designed for the study, and 3 replicates were set for each group of experiments. After 45 days of growth, the plants were harvested and analyzed.

[0043] Table 1 Experimental design of foliar spraying of six kinds of control agents

[0044]

[0045] 1.5 Determination of plant biomass and heavy metal content

[0046] (1) Plant biomass

[0047] After the samples were harvested, they were repeatedly rinsed with distilled water until no soil remained. The plants were harvested separately according to the aboveground and underground parts, then placed in an oven at 105°C for 2 hours, and then dried at 85°C to constant weight for subsequent dry weight determination.

[0048] (2) Determination of cadmium and divalent metal elements (Ca, Mg, Cu, Zn) content in plants and calculation of enrichment indexes

[0049] The dried plant samples were ground into powder and digested using the HNO3-HClO4 method. The Cd and divalent metal elements (Ca, Mg, Cu, Zn) in different parts were determined using an atomic absorption spectrophotometer (Perkin-Elmer, AA700, USA). The enrichment index formula is as follows:

[0050] Plant Cd content (HCC, mg kg-1) = whole plant Cd accumulation / whole plant dry weight (2-1)

[0051] BCF = plant Cd content / soil Cd content (2-2)

[0052] Transport coefficient (TF) = aboveground Cd accumulation / underground Cd accumulation (2-3)

[0053] Unit accumulation (BCQ, mg kg-1) = whole plant dry weight × plant Cd content / single pot soil weight (2-4)

[0054] 2 Results and analysis

[0055] The absorption and transport of cadmium in soil by annual ryegrass is affected by many factors, including some nutrients, the type and content of chelates, signal molecule regulation, etc. Therefore, we selected six types of cadmium transport inhibitors, namely NO, Se, Si, SAC, SA and S, with different concentrations to investigate their effects on the absorption and transport of cadmium by annual ryegrass.

[0056] 2.1 Effects of six foliar inhibitors on the biomass of annual ryegrass under cadmium stress

[0057] As the concentration of cadmium pollution increases, the annual ryegrass is increasingly stressed by cadmium, showing characteristics such as reduced biomass and shorter plant growth. Spraying the appropriate concentration of inhibitors on the leaves can effectively alleviate cadmium stress, thereby increasing the biomass of ryegrass. Figure 2 In general, under various concentrations of cadmium pollution, the aboveground dry weight of plants increased with the increase of the concentration of the six exogenous inhibitors, but the effects on the root dry weight were different. Compared with the control treatment, the aboveground dry weight and root dry weight increased significantly after the effects of NO, Se, and Si.

[0058] At 3 mg kg -1 Under cadmium pollution, high concentrations of NO (6 mg·L -1 ) treatment had the most significant promoting effect, with the dry weight of the aboveground part and roots increased by 18% and 23%, respectively, while the spraying of low concentration NO had a certain inhibitory effect on the dry weight of the aboveground part of the plant, which decreased by 12% compared with the control.

[0059] At 5 mg kg -1Under cadmium pollution, with the increase of Si spraying concentration, the aboveground and root dry weight of annual ryegrass gradually increased. -1 ) when the aboveground dry weight was 4.153 g, which was 30% higher than that without cadmium pollution. However, the effect of spraying NO on the biomass of ryegrass was slightly different from that of Si. When the spraying concentration was NO (4 mg·L -1 ) when the aboveground dry weight was 4.215g, which was 34% higher than that without cadmium pollution treatment.

[0060] 10mg kg -1 Under cadmium pollution, spraying high concentration of NO (6mg·L -1 ) had the best effect on promoting the biomass of ryegrass, with the dry weight of the aboveground part and the root being 4.310g and 2.181g respectively, which was 42% and 65% higher than that without any treatment of cadmium pollution. It can be seen that different types and concentrations of inhibitors have different effects on the absorption and transport of cadmium in annual ryegrass. -1 The NO treatment effect is more suitable for soils with low, medium and high cadmium pollution.

[0061] 2.2 Effects of foliar spraying of inhibitors on cadmium absorption and translocation in annual ryegrass

[0062] According to the National Food Safety Standard Limits of Contaminants in Food (GB2762-2017), the limit of cadmium in fresh leafy vegetables is 0.2 mg kg -1 (The moisture content of fresh annual ryegrass is 90%, and the limit value of cadmium in dry weight is 1.8 mg kg -1 ). Changes in plant cadmium content after different concentrations of cadmium stress are as follows Figure 3 shown.

[0063] Under various concentrations of cadmium stress, the cadmium content in the aboveground part of the annual ryegrass that was not treated with foliar barrier agents exceeded the standard. Foliar spraying of gradient concentrations of NO, Si, Se, and S can reduce the cadmium content in the aboveground part of the plant to varying degrees, and the cadmium reduction effect of the above four high-concentration barrier treatments is significantly better than that of low-concentration foliar barrier agents. However, the effects of SA and SAC showed opposite trends, and the cadmium content in the aboveground part increased slightly compared with the control. The specific performance is:

[0064] 3mg kg -1 Under cadmium pollution, with the increase of the concentration of exogenous NO, the cadmium content in the aboveground parts decreased by 55%, among which NO (6 mg·L -1 ) treatment had the best effect in reducing cadmium, with the cadmium content in the aboveground part being 0.183 mg kg -1 It is far lower than the limit value of cadmium in dry weight state (1.8mg·kg -1). However, the effect on the cadmium content in the underground part was somewhat different. After the low concentration NO treatment, the cadmium content in the underground part of the ryegrass reached a maximum of 49.876 mg kg -1 , only 3mg·kg -1 Pollution treatment increased by 56%. 5mg·kg -1 Under cadmium pollution, the cadmium content in the aboveground part of the plants in the high-concentration Si treatment was the lowest at 1.3 mg kg -1 Compared with the control, it was reduced by 74%. After spraying high concentration NO, the cadmium content in the aboveground part was 2.023 mg kg -1 , only 5mg·kg -1 Cadmium pollution was reduced by 64%, and the cadmium content in the roots was 65.881 mg kg -1 , compared with 5mg·kg -1 Cadmium pollution increased by only 34%.

[0065] 10mg kg -1 Under cadmium pollution, high concentrations of NO and Si had the most significant effect on reducing the aboveground cadmium concentration, with 5.707 mg kg -1 and 5.263 mg kg -1 , only 10mg·kg -1 Compared with the above, the levels of cadmium in the roots of annual ryegrass were reduced by 48% and 45%. This shows that without any treatment of cadmium pollution, the roots of annual ryegrass absorbed Cd and continuously transported it to the aboveground parts, causing the cadmium content in the aboveground parts to exceed the standard; foliar spraying of NO and Si reduced the ability of cadmium to be transported from the roots to the aboveground parts. At the same time, with the increase in spraying concentration, more cadmium was retained in the roots, enhancing the concentration effect of cadmium enrichment in the roots.

[0066] 2.3 Effects of six inhibitors on cadmium transport in annual ryegrass

[0067] like Figure 4 As shown in the figure, the six inhibitors sprayed on the leaves can reduce the cadmium transport coefficient of annual ryegrass to a certain extent. -1 SAC in cadmium-contaminated soil has an opposite trend to the other five foliar inhibitors. With the increase of exogenous SAC concentration, the TF value gradually increases.

[0068] At 3 mg kg -1 In the cadmium-contaminated soil, since the cadmium pollution level of the soil itself was relatively light, the use of six foliar inhibitors did not significantly reduce the cadmium transport coefficient of annual ryegrass. -1 In cadmium-contaminated soil, we can find high concentrations of NO (6 mg·L -1 ) and Si (70 mg·L -1) application significantly reduced the ability of cadmium transport from roots to aboveground parts, which was reduced by 115% and 240.2% respectively compared with the control. -1 In cadmium-contaminated soil, the lowest TF value was 0.08 when high concentration NO was applied, which was 268% lower than that of the control. It can be seen that under the three cadmium pollution concentrations, exogenous NO spraying can inhibit the transport of Cd from the roots to the aboveground parts of ryegrass, and the effect is best when applied at a high concentration.

[0069] 2.4 Effects of foliar spraying of three dominant inhibitors on plant essential elements and Cd absorption

[0070] The results of the previous study on the effects of six foliar inhibitors on the dry weight and cadmium absorption of annual ryegrass showed that the cadmium content in the aboveground parts of plants was lower under the three inhibitors, NO, Se and Si, and the TF was also lower. Therefore, we will further study the competitive relationship between cadmium transport and four metal cations in annual ryegrass under the application of the three inhibitors.

[0071] (1) Interaction between Cd stress and ion content in annual ryegrass

[0072] like Figure 5 To explore the sources of changes in ion composition under the action of three major exogenous inhibitors (NO, Se, Si), a comprehensive principal component analysis (PCA) was performed to analyze the changes in elements in different tissues. -1 Under the treatment concentration of , the cumulative contribution rate of PC1 and PC2 in leaves to the total variation reached 83.9%, of which PC1 accounted for 58.7% of the total variation, effectively distinguishing the treatment group from the control group; Ca and Zn contributed more to PC1, while Mg and Cu contributed more to PC2. The ion composition of the roots was slightly different from that of the leaves, of which PC1 and PC2 contributed more to 85.6%, and PC1 alone explained 58.2% of the total variation, clearly distinguishing the NO and Se treatment groups from the control group; Ca, Mg and Zn contributed significantly to PC1, while Zn and Cu contributed more to PC2.

[0073] At 5 mg kg -1 Under the treatment concentration of , the cumulative contribution rate of PC1 and PC2 of leaves to the total variation was 81.7%, PC1 explained 58.9% of the total variation, and successfully distinguished the Se and NO treatment groups from the control group; Zn, Mg and Cu contributed significantly to PC1, and Ca was the main contributing element of PC2. The ion composition of the roots showed that the cumulative contribution rate of PC1 and PC2 was 78.5%, PC1 was responsible for 46% of the total variation, and distinguished the three inhibitor treatment groups from the control group; Ca and Zn mainly contributed to PC1, while Mg and Cu mainly contributed to PC2.

[0074] At 10 mg kg -1 Under the treatment concentration of , the cumulative contribution rate of PC1 and PC2 in leaves was as high as 86.78%, and PC1 alone explained 70.7% of the total variation, clearly distinguishing the NO treatment group from the control group; Zn and Ca were the main contributing elements to PC1, and Mg and Cu mainly contributed to PC2. In contrast, the cumulative contribution rate of the ion composition of the root in PC1 and PC2 was 43.5%, and the distinction between the NO treatment and the control group was significant; Ca and Zn made a greater contribution to PC1, while Mg and Cu contributed significantly to PC2.

[0075] (2) Correlation analysis of metal element contents in the aboveground parts of annual ryegrass

[0076] like Figure 6 As shown in the figure, after spraying NO inhibitor on leaves, 3 mg kg -1 Cadmium pollution treatment Cd and Ca, Mg, Cu, Zn synergistically showed a very significant positive correlation (P < 0.01); 5 mg·kg -1 In the cadmium pollution treatment, Cd and Mg, Zn synergized with each other, showing a very significant positive correlation (P<0.01), while the synergistic relationship with Ca and Cu turned into antagonism, showing a very significant negative correlation (P<0.01); 10 mg·kg -1 In the cadmium pollution treatment, Cd was synergistic with Ca, Cu, and Zn, showing a significant positive correlation (P<0.05), while it was extremely significantly negatively correlated with Mg (P<0.01). In general, the positive correlation between Cd and a large number of elements was mainly observed in leaves (P<0.05), indicating that NO treatment promoted the absorption of essential elements by the roots, thereby affecting the accumulation of these elements in the leaves.

[0077] After spraying Se blocking agent on leaves, 3mg·kg -1 In the cadmium pollution treatment, the synergistic relationship between Cd and Ca and Cu was transformed into antagonism (P<0.01), and it was positively correlated with Mg and Zn (P<0.05). -1 There was a very significant positive correlation between Cd and the four elements in cadmium-contaminated soil.

[0078] After foliar spraying of Si inhibitor, 3mg·kg -1 Cadmium pollution treatment Cd is antagonistic to Ca, Mg, Cu, and Zn and is negatively correlated; 5, 10 mg·kg -1In the cadmium pollution treatment, Cd was also significantly negatively correlated with the four divalent elements (P<0.05); the negative correlation between Cd and the macroelements was mainly observed in the leaves (P<0.05), indicating that Si treatment inhibited the absorption of essential elements by the roots, thereby affecting the accumulation of these elements in the leaves. Cd was significantly negatively correlated with the four elements. The results showed that there was a significant inhibitory transport effect between Cd and the four elements in the leaves. It is speculated that these four essential elements may be accumulated in the roots of annual ryegrass, blocking Cd from entering the leaves. The root system is well developed and the metabolism is vigorous. The roots, stems, and leaves need to absorb a large amount of various elements and reuse them during the rapid development process. The Cd entering each organ significantly disturbed the element metabolism process of the leaves.

[0079] 3 Summary

[0080] (1) Different types and concentrations of inhibitors have different effects on cadmium absorption and transport in annual ryegrass. -1 The NO treatment effect was 3, 5 and 10 mg·kg -1 Cadmium-contaminated soil is more suitable. -1 In cadmium-contaminated soil, when the concentration of Si (70 mg·L -1 ), the maximum dry weights of the aboveground and underground parts were 4.153g and 2.120g respectively.

[0081] (2) Under three cadmium pollution concentrations, 3 and 5 mg kg -1 Cadmium pollution has been controlled by leaf surface treatment, and the cadmium content in the aboveground part has reached the national safety standard. -1 The high concentration of NO treatment had the best effect in reducing cadmium, and the cadmium content in the aboveground part was 0.183 mg·kg -1 It is far lower than the limit value of cadmium in dry weight state (1.8mg·kg -1 ); at 5 mg kg -1 The lowest cadmium content in the aboveground part of the high concentration Si treatment was 1.3 mg kg -1 Compared with the control, it was reduced by 74%. -1 When NO(6mg·L -1 ) treatment had the most significant effect on reducing the cadmium concentration in the aboveground parts, which was reduced by 48% to 2.63 mg·kg -1 Under three cadmium pollution concentrations, exogenous NO spraying could inhibit the transport of Cd from the roots to the aboveground parts of ryegrass, and the effect was best when applied at a high concentration.

[0082] (3) The application of three dominant inhibitors (NO, Si and Se) interfered with the variation of key divalent ion groups in annual ryegrass, and the variation of ion groups was more obvious after NO was applied to the roots of annual ryegrass. NO treatment promoted the absorption of essential elements by the roots, which in turn affected the accumulation of these elements in the leaves. The results showed that the positive correlation between Cd and macroelements (Ca, Mg, Cu, Zn) was mainly observed in the leaves (P<0.05).

[0083] Example 2

[0084] 1 Materials and methods

[0085] 1.1 Material preparation

[0086] The materials of this embodiment are selected based on Example 1, selecting annual ryegrass as the root enrichment plant, selecting NO as the foliar barrier agent, and selecting a microbial combination (Bacillus subtilis: Citrobacter rodentium: Bacillus cereus = 20%: 20%: 10%) as the microbial composite agent.

[0087] 1.2 Soil sample collection

[0088] Rhizosphere soil samples: After 45 days of annual ryegrass stress, the annual ryegrass was gently pulled up, and the roots were shaken to remove large pieces of soil, and 10 mg kg -1 The rhizosphere soil under cadmium stress and blank soil without plants were passed through a 20-mesh sieve and then quickly frozen in liquid nitrogen. Finally, the collected samples were stored at -80°C. Each experimental group contained 3 biological replicates.

[0089] 1.3 Experimental design

[0090] As shown in Table 2, the concentration of NO applied was: NO at 3 mg kg -1 The application concentration in cadmium-contaminated soil was set at 6 mg·L -1 ; 5mg·kg -1 The application concentration in cadmium-contaminated soil was set at 30 mg·L -1 ; 10mg·kg -1 The application concentration in cadmium-contaminated soil was set at 30 mg·L -1 .

[0091] Table 2 Experimental design for the construction of a cadmium enrichment enhancement system for plant roots under the action of NO

[0092]

[0093] Exogenous application of NO in different parts to block cadmium transport: When the annual ryegrass plants grow to 45 days, the microbial combination screened in the early stage of this experiment (Bacillus subtilis, Bacillus cereus and Citrobacter inoculated with 20%, 10% and 20% respectively) was injected into the root layer of the plant through a syringe, and the five-point inoculation method was used to ensure that the bacterial solution was evenly distributed in different positions. Injection was performed every 7 days, 20 ml each time, and a total of 4 injections were performed. For a single pot of annual ryegrass, a total of 80 ml of bacterial solution was injected. The application of NO is divided into foliar spraying and direct soil application. The method and inoculation amount of NO in the soil are the same as the microbial combination bacterial solution inoculation, and the method of foliar spraying of NO is the same as 4.1.4. When NO and the microbial combination are applied together, the microbial solution is inoculated first, and the NO control treatment is performed the next day. A total of 18 groups of experiments were designed in this study, each with 3 replicates. After a total growth of 45 days, the plants were harvested for analysis.

[0094] 1.4 Test methods and data processing

[0095] The annual Cd removal rate formula of annual ryegrass is as follows:

[0096]

[0097] In formula 5-1, BCQ is the Cd accumulation per unit soil weight, and the growth cycle of annual ryegrass is one year with four harvest seasons.

[0098] 1.5 Analysis of soil microbial community composition

[0099] use Total microbial DNA was extracted using a DNA isolation kit (Omega Bio-tek Inc., Doraville, GA, USA). Initially, an initial denaturation of 3 minutes was performed at 95°C, followed by 35 cycles, each cycle including 30 seconds at 95°C, 30 seconds at 55°C, 45 seconds at 72°C, and finally a 10-minute extension phase at 72°C. Afterwards, the amplified fragments were recovered from a 2% agarose gel and purified using an AxyPrep DNA gel extraction kit (AxyPrep Biosciences, Union City, CA, USA), followed by quantitative analysis using QuantiFluorTM-ST (Promega, USA).

[0100] First, the raw data (Rawdata) obtained by sequencing was processed, and the double-end data was spliced ​​using the overlap method, and quality control and chimera filtering were performed on it to obtain high-quality CleanData. Subsequently, the concept of ASVs (Amplicon Sequence Variants) was used to construct the class OUT (Operational Taxonomic Units). Amplicon sequence variations (ASVs) were clustered with a similarity cutoff of 100%, and chimera filtering and ASV clustering were performed simultaneously using UPARSE (Version 7.1 http: / / drive5.com / uparse / ). Next, the feature abundance in each sample was normalized according to the SILVA classifier. Subsequently, the Blast tool was used for sequence alignment, and the feature sequence was annotated for each representative sequence using the SILVA database. Finally, the ASV feature table and feature sequence were obtained for subsequent diversity analysis and other work.

[0101] 2 Results and analysis

[0102] Under three cadmium pollution conditions, we conducted five treatments, namely, microbial treatment only, foliar spraying of NO, foliar spraying of NO + microorganisms, soil application of NO, and soil application of NO + microorganisms, on the effects of the cadmium enrichment construction system in the roots of annual ryegrass. The specific research results are as follows:

[0103] 2.1 Effects of NO application on biomass and cadmium content of annual ryegrass at different locations

[0104] like Figure 7 As shown in the figure, through the screening of six foliar inhibitors in the early stage, we found that the exogenous application of NO at 3, 5, and 10 mg kg -1 The cadmium reduction effect on the upper part of annual ryegrass in cadmium-contaminated soil was good, so we selected NO as the dominant inhibitor to further investigate the effect of different application sites on cadmium enrichment in annual ryegrass.

[0105] At 3 mg kg -1 In cadmium-contaminated soil, the four control treatments can increase the dry weight of the upper and underground parts of annual ryegrass to a certain extent compared with the control, but the difference between the four control treatments is not significant. In addition, different control treatments can make the upper part of annual ryegrass meet the national edible standard. -1 The lowest cadmium content in aboveground parts was 0.420 mg kg when NO and microorganisms were combined. -1 , which was 84.7% lower than the control, and the cadmium content in the roots increased by 45.1% compared with only adding microorganisms and 97.8% compared with the control.

[0106] At 5 mg kg -1 In cadmium-contaminated soil, 30 mg·L -1 The combined application of NO and microorganisms made the annual ryegrass aboveground dry weight reach the maximum, increasing by 16.7% compared with the microorganism treatment alone. In addition, all four control treatments made the annual ryegrass meet the national edible standard, among which the foliar spraying of 30 mg·L -1 NO combined with microorganisms and soil application 30 mg·L -1 The combined application of NO and microorganisms reduced the cadmium content in the aboveground parts by 0.320 mg·kg -1 and 0.410 mg kg -1 The cadmium content in the roots increased by 42.3% compared with the addition of microorganisms only and increased by 112.0% compared with the control; it increased by 35.8% compared with the addition of microorganisms only and increased by 102.4% compared with the control.

[0107] At 10 mg kg -1 In cadmium-contaminated soil, 30 mg·L -1 The aboveground and root dry weights of annual ryegrass treated with NO and microorganisms increased by 25.0% and 87.5%, respectively, compared with the control treated with only microorganisms. -1 The cadmium contents in the aboveground parts after NO and NO+ and microbial control treatments reached the standards of 1.503 and 1.827 mg·kg, respectively. -1 The lowest cadmium content in the aboveground part of the control treatment with the combined application of the two was 1.503 mg kg -1 The highest cadmium content in the root was 92.616 mg kg -1 , an increase of 42.3% compared with the addition of microorganisms only and an increase of 112.0% compared with the control.

[0108] 2.2 Effects of NO application at different sites on cadmium accumulation in the lower part of annual ryegrass

[0109] like Figure 8 As shown in the figure, under three cadmium pollution concentrations, the five treatments can increase the cadmium accumulation in the lower part of the annual ryegrass to varying degrees. In addition, the cadmium accumulation in the roots under the action of microorganisms is higher than that in the treatment using NO alone. -1 The combined application of NO foliar spraying and microorganisms can make the cadmium accumulation in the lower part of annual ryegrass reach the maximum value of 0.128 and 0.118 mg, respectively, which is 106.5% and 78.8% higher than that of the microbial combination treatment alone, and 180% and 76.9% higher than that of the control.

[0110] The accumulation patterns of NO in the roots of annual ryegrass were different for different NO application sites. Under low-concentration cadmium pollution, soil application of NO increased cadmium enrichment in the roots of annual ryegrass more significantly than foliar spraying of NO. However, under high-concentration cadmium pollution, at 30 mg·L -1 Foliar spraying of NO was better than soil application. At the same time, for the annual ryegrass-microorganism remediation system, foliar spraying of NO was more effective than soil application under three cadmium pollution concentrations.

[0111] It can be seen that the combination of foliar spraying of NO and microorganisms is an effective measure to enhance the cadmium enrichment in the roots of annual ryegrass. Under low-concentration cadmium pollution conditions, the optimum concentration of NO is 8 mg·L -1 Under the conditions of medium and high concentrations of cadmium pollution, the optimum concentration of NO is 30 mg·L -1 On the one hand, this treatment method has a significant trend of reducing the cadmium content in the aboveground part, and the cadmium content in the aboveground part of the annual ryegrass grass under the three cadmium pollution concentrations all reached the national standard limit. In addition, through this combined action method, more cadmium was fixed in the roots of the plants, while the efficiency of plant extraction and cadmium repair was improved.

[0112] 2.3 Effects of NO application at different sites on the annual restoration efficiency of annual ryegrass

[0113] The effect of the construction of the root cadmium enrichment and enhancement system on the biomass and Cd content of annual ryegrass is ultimately reflected in the annual removal rate. We calculated the annual Cd removal rate of the root cadmium enrichment and enhancement system under the assumption that the annual growth cycle of annual ryegrass is four seasons. The annual Cd removal rate of different treatments is shown in Table 3. BCQ directly affects the annual removal rate of Cd. Changes in Cd content and plant biomass lead to continuous changes in BCQ. Under three cadmium pollution concentrations, the combined application of foliar spraying of NO and microbial combination maximized the BCQ of annual ryegrass. Overall, the five treatments all have an improving effect on the annual Cd removal rate of annual ryegrass.

[0114] At 3 mg kg -1 Under cadmium pollution conditions, microbial treatment or 8 mg·L -1 The application of NO significantly increased the annual removal rate of Cd. The annual removal rate of Cd by foliar spraying of NO was greater than that by soil application of NO, which was 30.5% and 33.5% respectively. In addition, the combined application of microorganisms and NO also had a certain promoting effect on the enrichment system. Foliar spraying of 8 mg·L -1 The combined application of NO and microorganisms increased NO by 58.1% compared with the treatment with microorganisms alone; soil spraying of 8 mg·L -1 The combined application of NO and microorganisms increased NO by 39.6% compared with the microorganism treatment alone.

[0115] At 5 mg kg -1 Under cadmium pollution conditions, 30 mg·L -1 The combined application of NO and microorganisms had the highest removal rate, reaching 24.2%, which was 131% higher than that of microbial treatment alone. -1 Compared with no treatment, the increase was 172%. -1 Under cadmium pollution conditions, 30 mg·L -1 The combined application of NO and microorganisms had the highest clearance rate, reaching 11.8%, which was comparable to 10 mg kg -1 Compared with no treatment, it increased by 268.8%. -1 Under cadmium pollution conditions, we found that 10 mg kg -1 Under pollution conditions, higher concentrations of NO can be added to observe whether its effect will be better.

[0116] In summary, similar to the changing trends of biomass and Cd content, more available Cd in the soil was activated when Bacillus was exogenously inoculated in the rhizosphere, and the application of NO to the soil may have reduced the cadmium accumulated in the roots of plants and caused a partial antagonistic reaction with microorganisms.

[0117] Table 3 Changes in annual Cd removal rate under root cadmium enrichment enhancement system

[0118]

[0119] Table 3 Changes in annual Cd removal rate under root cadmium enrichment enhancement system

[0120]

[0121] 3 Summary

[0122] (1) Foliar spraying of NO, soil application of NO, foliar spraying of NO+microorganisms, and soil application of NO+microorganisms can increase the dry weight of annual ryegrass to varying degrees. Under the three cadmium pollution conditions, foliar spraying of NO and microorganisms combined application had the best effect on reducing cadmium in the aboveground part of annual ryegrass, reaching the national safety standard for use, and the root cadmium enrichment effect was also the best. Among them, at 3 mg kg -1 Under cadmium pollution, 8 mg·L -1 The lowest cadmium content in the aboveground part of the plant was 0.420 mg kg when NO and microorganisms were combined. -1 The cadmium content in the roots increased by 45.1% compared with the addition of microorganisms alone and by 97.8% compared with the control. -1 Under cadmium pollution conditions, 30 mg·L was sprayed on the leaves -1 NO combined with microorganisms and soil application 30 mg·L-1 The combined application of NO and microorganisms reduced the cadmium content in the aboveground parts by 0.32 mg·kg -1 and 0.41 mg kg -1 , the cadmium content in the roots increased by 42.3% and 35.8% respectively compared with the addition of microorganisms alone. -1 Under cadmium pollution conditions, 30 mg·L -1 The cadmium content in the aboveground part of the NO and microbial control treatment reached the lowest level of 1.503 mg kg -1 The cadmium content in the roots was the highest, increasing by 42.3% compared with the addition of only microorganisms.

[0123] (2) Under the three cadmium pollution concentrations, the BCQ of annual ryegrass was maximized by the combination of foliar spraying of NO and microbial combination. -1 Under cadmium pollution conditions, the removal rate of NO-30L+m treatment was the highest, reaching 13.4%, which was 131% higher than that of microbial treatment alone and 5 mg kg -1 Compared with no treatment, the increase was 172%. -1 Under cadmium pollution conditions, NO-30L+m treatment also had the highest removal rate, reaching 11.2%.

[0124] The above embodiments describe only the basic principles, main features and / or advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and the invention content of the specification only describe the principles or specific cases of the present invention. Without departing from the essence of the innovative idea of ​​the present invention, the innovative scheme of the present invention may be subject to various changes and improvements, and these changes and improvements all fall within the scope of protection required by the present invention.

Claims

1. A method for controlling cadmium transport in hyperaccumulator plants, characterized in that: Annual ryegrass was grown in soil containing cadmium and foliar control agents were applied to the annual ryegrass.

2. The method for controlling cadmium transport in hyperaccumulator plants according to claim 1, characterized in that: The foliar barrier agent is any one or more of sodium selenite, NaHS hydrate, sodium nitroprusside, S-allyl-L-cysteine, sodium silicate and salicylic acid.

3. The method for controlling cadmium transport in hyperaccumulator plants according to claim 2, characterized in that: The foliar barrier agent is sodium nitroprusside.

4. The method for controlling cadmium transport in hyperaccumulator plants according to claim 3, characterized in that: The foliar control agent is applied by foliar spraying or root injection, any one or more of the following.

5. The method for controlling cadmium transport in hyperaccumulator plants according to claim 3, characterized in that: A microbial compound is also added to the roots.

6. The method for controlling cadmium transport in hyperaccumulator plants according to claim 5, characterized in that: The microbial composite bacterial agent comprises: 15-25% of Bacillus subtilis, 5-15% of Bacillus cereus and 15-25% of Citrobacter acidophilus.

7. The method for controlling cadmium transport in hyperaccumulator plants according to claim 6, characterized in that: The microbial composite agent comprises: 20% of Bacillus subtilis, 10% of Bacillus cereus and 20% of Citrobacter acidophilus.

8. The method for controlling cadmium transport in hyperaccumulator plants according to any one of claims 1 to 7, characterized in that: The cadmium content in the soil is 0.1-10 mg·L -1 .

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