Application of composite action of microbial agent and charcoal in improvement of cadmium resistance of plants

Abstract

According to the method disclosed by the invention, arbuscular mycorrhizal fungi and biochar are added while multiple-leaf goldenrain trees are planted in cadmium-polluted soil. Compared with a traditional remediation mode of independently utilizing plants or microorganisms or biochar, the microorganism-plant-biochar combined remediation has the advantages that the plants, the microorganisms and the biochar are co-cultured to form a symbiotic system, so that the cadmium content in the soil is remarkably reduced, the remediation capacity of the plants is enhanced, and meanwhile, the growth of the plants is remarkably improved. The strategy breaks through the limitation that in the prior art, only improvement of plant resistance is concerned, and the strategy more focuses on the double effects of improving the phytoremediation capacity and promoting the growth effect, so that the maximization of the phytoremediation effect is achieved.

Description

[0001] The invention relates to the technical field of heavy metal pollution remediation, and in particular to a method for promoting Koelreuteria bipinnata to remediate soil cadmium pollution. Background Art

[0002] In recent years, with the rapid development of industrialization and urbanization, cadmium (Cd) pollution has become a widespread ecological and environmental problem in southern China. Some studies have begun to explore the use of phytoremediation technology to treat cadmium pollution in soil, by selecting and cultivating specific plants to absorb or fix cadmium to reduce its harm to the environment and organisms.

[0003] Environmental remediation using plants is not perfect. Traditional phytoremediation methods rely on the natural growth of plants and their absorption or passivation of heavy metals. However, this process is usually very slow and takes years or even longer to observe obvious remediation effects. For heavily polluted areas that are in urgent need of treatment, this method obviously cannot meet the needs of rapid response. Many species used for phytoremediation can only tolerate mildly or moderately polluted environments, and their biomass may be significantly reduced or even die under high concentrations of heavy metals. This greatly limits their scope of application.

[0004] Phytoremediation mainly reduces the bioavailability of heavy metals by fixing them in roots or converting them into less toxic forms, but this does not mean that these pollutants are completely removed from the environment. Therefore, once conditions change (such as pH value, redox potential, etc.), the fixed heavy metals may still be released back into the environment. Therefore, the use of phytoremediation technology alone is often difficult to work, and must be combined with other treatment methods to achieve a stable and efficient bioremediation effect. Summary of the invention

[0005] Koelreuteria paniculata Koelreuteria bipinnata ) As an economic tree species, it not only has a certain cadmium resistance ability, but also has medicinal value. However, relying solely on plants themselves is not enough to achieve an ideal restoration effect. The present invention introduces Koelreuteria paniculata with clump-planted mycorrhizal fungi and biochar, which can enhance the bioremediation of soil cadmium pollution by Koelreuteria paniculata and reduce the toxic effects of cadmium on plants.

[0006] The object of the present invention is to provide a method for reducing the cadmium content in soil; The above-mentioned purpose of the present invention is achieved through the following technical solutions: A method to reduce soil cadmium levels by planting cadmium-tolerant plants in cadmium-contaminated soils and adding arbuscular mycorrhizal fungi and biochar to cadmium-contaminated soils.

[0007] The cadmium-tolerant plant is Koelreuteria bipinnata; The arbuscular mycorrhizal fungi are root cysts of endophytic fungi, inoculated surface cysts of diverse fungi, or a mixture of the two; Preferably, the mass ratio of the arbuscular mycorrhizal fungi to the matrix soil is 1:45; The biochar is prepared by pyrolyzing rice husk, an agricultural waste, at 550° C. for 2 hours under anoxic conditions; Preferably, the amount of biochar added is 3% of the mass of the substrate soil; Preferably, in a low cadmium environment (15 mg / kg), a combination of Rhizosporium endoradiatum and biochar was selected; Preferably, under a high cadmium environment (150 mg / kg), a combination of intraradical Rhizospora, surface diverse Rhizospora and biochar is selected.

[0008] The present invention has the following beneficial effects: (1) Compared with the traditional restoration method of using plants, microorganisms or biochar alone, the microorganism-plant-biochar combined restoration of the present invention significantly reduces the effective cadmium concentration in the soil by co-cultivating plants with microorganisms and biochar to form a symbiotic system.

[0009] (2) The combination of arbuscular mycorrhizal fungi and biochar enhanced the plant enrichment under cadmium stress, while reducing the cadmium content in various plant tissues and organs, thereby reducing the cadmium toxicity.

[0010] (3) While reducing the cadmium content in the soil, the present invention selects different combinations according to different degrees of soil cadmium pollution. In a low cadmium environment (15 mg / kg), the combination of Rhizocystis endoradiata and biochar is preferred; in a high cadmium environment (150 mg / kg), the combination of Rhizocystis endoradiata, Polycystis terrestris and biochar is preferred. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 :Effects of arbuscular mycorrhizal fungi and biochar on the net photosynthetic rate of Koelreuteria bipinnata.

[0012] Figure 2 :Effects of arbuscular mycorrhizal fungi and biochar on water use efficiency of Koelreuteria bipinnata.

[0013] Figure 3 :Effects of arbuscular mycorrhizal fungi and biochar on the total biomass of Koelreuteria bipinnata.

[0014] Figure 4 :Effects of arbuscular mycorrhizal fungi and biochar on the root-to-shoot ratio of Koelreuteria paniculata.

[0015] Figure 5 :Effects of arbuscular mycorrhizal fungi and biochar on N of Koelreuteria bipinnata.

[0016] Figure 6 :Effects of arbuscular mycorrhizal fungi and biochar on P of Koelreuteria bipinnata.

[0017] Figure 7 :Effects of arbuscular mycorrhizal fungi and biochar on N:P of Koelreuteria bipinnata.

[0018] Figure 8 :Effects of arbuscular mycorrhizal fungi and biochar on cadmium contents in aboveground and underground parts of Koelreuteria bipinnata.

[0019] Fig. 9 : Effects of arbuscular mycorrhizal fungi and biochar complex on total cadmium in soil.

[0020] Fig.10 :Effects of arbuscular mycorrhizal fungi and biochar on DTPA available cadmium content in soil.

[0021] Fig.11 :Effects of arbuscular mycorrhizal fungi and biochar on the cadmium enrichment coefficient of Koelreuteria bipinnata.

[0022] Fig.12 :Effects of arbuscular mycorrhizal fungi and biochar on the transfer coefficient of Koelreuteria bipinnata. DETAILED DESCRIPTION

[0023] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form unless otherwise specified. The reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Example

[0024] S1. Acquisition of arbuscular mycorrhizal fungi; S1.1. Select two arbuscular mycorrhizal fungi; The two species of bacteria selected in the present invention are ground surface diverse cysts ( Diversispora versiformis ,(P.Karst.) Oehl, GA Silva & Sieverd.(BGC GD01C), referred to as Dv)) and Rhizocystis endoradici ( Rhizophagus intraradices , (NC Schenck & GS Sm.) C. Walker & A. Schüßler (BGC BJ09), referred to as Ri), all provided by the Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry Sciences; S1.2, propagate arbuscular mycorrhizal fungi; Both mycorrhizal fungi used sorghum as the host plant and autoclaved fine sand as the substrate for 5 months of propagation culture; S1.3, obtain the expanded arbuscular mycorrhizal fungi; After the cultivation, the arbuscular mycorrhizal fungi inoculant was obtained, including spores, hyphae, and some infected root segments. The surface diverse cysts and the root cysts were propagated by the above method. S1.4, obtain sterilized arbuscular mycorrhizal fungi; The arbuscular mycorrhizal fungi obtained above are sterilized by high temperature and high pressure to obtain sterilized arbuscular mycorrhizal fungi; S2. Obtaining biochar; Rice husk biochar was purchased from Zhejiang Yangtze River Delta Junong Technology Development Co., Ltd. and was obtained by pyrolysis of agricultural waste rice husk at 550 °C for 2 h under anoxic conditions.

[0025] The basic properties of rice husk biochar are: pH 8.1, total nitrogen 3.92 g kg -1 , available phosphorus 0.29 g kg -1 , total carbon 35.2%, ash 33.18%, sulfur 0.02%, specific surface area 103.12 m 2 g -1 , and then sterilized by 25kGy gamma ray radiation for later use.

[0026] S3, plant the Koelreuteria paniculata in a flower pot; S3.1, seedling treatment; One-year-old Koelreuteria compound pinnate seedlings were used as research materials. After sterilization and standardization to a uniform size, they were transplanted. Two seedlings were planted in each pot, for a total of 240 seedlings.

[0027] S3.2, transplanting seedlings; The seedlings were transplanted into pots with different treatments of arbuscular mycorrhizal fungi and biochar.

[0028] A 3 × 4 × 2 factorial design was used, including three levels of Cd stress treatment (0, 15, 150 mg kg -1 ), 4 levels of arbuscular mycorrhizal fungi inoculation treatments (inoculation with sterilized fungi, inoculation with root cysts, inoculation with surface cysts, and inoculation with a mixture of these two fungi) and 2 levels of biochar addition treatments (no addition or addition of biochar), a total of 24 treatments, 5 replicates per treatment, a total of 120 pots. The experimental potting matrix soil was Lin'an local soil (passed through a 2 mm sieve) and was sterilized by 25 KGy of gamma rays before use to eliminate the original indigenous microorganisms. The matrix soil in the pot was 1.8 kg.

[0029] The specific experimental groups are: CK group: inoculated with 40 g of sterilized mixed bacteria (equal proportions of Rhizospora intraradicata and Diversospora subterranea); Group B: 3% biochar added; Ri group: inoculated with 40 g of Rhizospora intraradicata; Dv group: inoculated with 40 g of Diversocystis terrestris; Ri+Dv group: inoculated with 40 g of mixed bacteria (equal proportions of Rhizospora intraradicata and Diversospora subterranea); Ri+B group: inoculated with 40 g of Rhizospora endophyta and added with 3% biochar; Dv+B group: inoculated with 40 g of Diversocystis sp. and added with 3% biochar; Ri+Dv+B group: 40 g of mixed bacteria (equal proportions of Rhizospora intraradicata and Diversospora subterranea) were inoculated and 3% biochar was added; S3.3, adding balancing solution; Add 5 mL of balancing solution (prepared with non-sterile mixed bacteria and sterilized distilled water in a ratio of 1:3) to the sterilized control treatment pot to adjust the microbial flora. Pass the balancing solution through a 400-mesh standard sieve and repeat three times to maintain the consistency of the microbial community in all pots except for arbuscular mycorrhizal fungi. S3.4, exogenous addition of CdCl2 solution; After 60 days of cultivation, the transplanted seedlings were treated with exogenous CdCl2 solution. Each group was treated with three different levels of cadmium (0, 15, and 150 mg kg -1 ); S3.5. Harvesting of Koelreuteria paniculata.

[0030] The potted plants were harvested after 1 year and 6 months, and the relevant indicators of the plants were measured.

[0031] Data statistical analysis was performed using R 4.2.1 (R Core Team, 2022) for three-way ANOVA. The experimental data were tested for normality and homogeneity of variance. Each data represents the mean ± standard error, and the LSD method was used to test the significance of the difference between different treatment combinations. The data marked with the same letter indicated that the difference was not significant after the LSD test (p>0.05).

[0032] Performance Testing 1. Determine the effects of the combined effects of arbuscular mycorrhizal fungi and biochar on the net photosynthetic rate and water use efficiency of Koelreuteria paniculata.

[0033] Three seedlings with relatively consistent growth were selected from each treatment. The net photosynthetic rate and water use efficiency of the leaves were measured using a Li-6400 photosynthetic meter (LI-COR, Lincoln, USA) from 8:00 to 11:30 on sunny days. The results are shown in the figure. Figure 1 and Figure 2In the accompanying drawings of this embodiment, all legends are expressed as follows: CK is inoculated with sterilized bacteria; B is added with biochar; Ri is inoculated with root cysts; Dv is inoculated with surface cysts; Ri+Dv is inoculated with a mixed strain of root cysts and surface cysts; Ri+B is added with root cysts and biochar; Dv+B is added with surface cysts and biochar; Ri+Dv+B is added with root cysts and surface cysts and biochar at the same time.

[0034] from Figure 1 It can be seen that at 0 mg kg -1 Under the cadmium treatment, whether adding arbuscular mycorrhizal fungi or adding biochar or the combination of biochar and arbuscular mycorrhizal fungi, the net photosynthetic rate of Koelreuteria bipinnata was significantly improved. -1 Under the cadmium treatment, the addition of Rhizocystis endophyticum or Diversicystis endophyticum or the combination of Rhizocystis endophyticum, Diversicystis endophyticum and biochar can significantly increase the net photosynthetic rate of Koelreuteria bipinnata. -1 The net photosynthetic rate of Koelreuteria bipinnata under cadmium treatment showed the same trend as that of 15 mg kg -1 Cadmium treatment.

[0035] from Figure 2 It can be seen that at 0 mg kg -1 Under the cadmium treatment, the combination of Rhizospora intraradicata and biochar can significantly improve the water use efficiency of Koelreuteria bipinnata. -1 Under the cadmium treatment, the addition of biochar alone can significantly improve the water use efficiency of Koelreuteria bipinnata. -1 Under cadmium treatment, adding ground-based diverse cyst molds alone can significantly improve the water use efficiency of Koelreuteria paniculata.

[0036] 2. Determine the effects of the combined effects of arbuscular mycorrhizal fungi and biochar on the total biomass and root-to-shoot ratio of Koelreuteria paniculata.

[0037] When harvesting, remove the plants from the soil; wash and dry the seedlings; use pruning shears to separate them into three parts: roots, stems, and leaves; place them in a 65℃ oven for 72 hours to dry until constant weight; measure the dry weight of roots, stems, and leaves, and calculate the total dry weight and root-to-crown ratio. Figure 3 and Figure 4 shown.

[0038] It can be seen that when no arbuscular mycorrhizal fungi or biochar were added, the biomass of Koelreuteria paniculata decreased significantly with the increase of cadmium stress; after adding arbuscular mycorrhizal fungi and / or biochar, the biomass of Koelreuteria paniculata increased significantly. Among them, under low cadmium environment, the combination of intraradical Rhizospora and biochar had the most significant effect in reducing the root-to-crown ratio of Koelreuteria paniculata; under high cadmium stress, the combination of intraradical Rhizospora, surface diverse cysts and biochar had the most significant effect in increasing the biomass of Koelreuteria paniculata.

[0039] 3. Determination of nitrogen, phosphorus and cadmium content in plant tissues.

[0040] The plant samples dried for 72 hours were ground with a sample grinder and passed through a 100-mesh standard sieve for the determination of total nitrogen, total phosphorus and cadmium in the plant's aboveground and root systems. Three replicates were taken for each treatment. The nitrogen content of plants was determined by sulfuric acid-hydrogen peroxide digestion using an AA3 continuous flow analyzer; The phosphorus content of plants was determined by the sulfuric acid-hydrogen peroxide method and the molybdenum antimony colorimetric method. The cadmium content in plants was determined by nitric acid-perchloric acid method.

[0041] Plant N content, plant P content, plant N: P and plant cadmium content are as follows Figure 5 , Figure 6 , Figure 7 and Figure 8 As shown; from Figure 5 It can be seen that at 0 mg kg -1 and 15 mg kg -1 Under the cadmium treatment, whether adding arbuscular mycorrhizal fungi or biochar or the combination of biochar and arbuscular mycorrhizal fungi can increase the N content of Koelreuteria bipinnata.

[0042] from Figure 6 It can be seen that the addition of arbuscular mycorrhizal fungi and / or biochar can significantly increase the P content of Koelreuteria bipinnata under cadmium stress, among which the combination of Rhizosporium endoradiatum and biochar has the most significant effect.

[0043] from Figure 7 It can be seen that at 15 mg kg -1 and 150 mg kg -1 Under the cadmium treatment, whether adding arbuscular mycorrhizal fungi or biochar or the combination of biochar and arbuscular mycorrhizal fungi can significantly reduce the N:P of Koelreuteria bipinnata.

[0044] from Figure 8It can be seen that under low cadmium stress, the combination of intraradical Rhizospora and biochar significantly reduced the cadmium content in the aboveground part of the plant, while also reducing the cadmium content in the plant root system; under high cadmium stress, adding a combination of arbuscular mycorrhizal fungi and biochar can significantly reduce the cadmium content in the aboveground part of Koelreuteria paniculata, among which the combination of intraradical Rhizospora, surface diverse cysts and biochar had the most significant effect.

[0045] 4. Determination of soil cadmium content.

[0046] After the soil was sieved through a 2 mm sieve to remove larger soil particles, the total cadmium and DTPA effective cadmium contents in the soil were determined, with three replicates for each treatment. The method for determining the total cadmium content in the soil was to use aqua regia-perchloric acid digestion and direct determination using flame atomic absorption spectrometry. The method for determining the effective cadmium content in plants was to use an atomic absorption spectrophotometer after extraction with a DTPA extractant. The results are shown in Figure 2. Fig. 9 and Fig.10 shown.

[0047] As can be seen from the figure, with the increase of cadmium stress, the total cadmium content in the soil increased significantly. After adding arbuscular mycorrhizal fungi and / or biochar, the total cadmium DTPA effective cadmium content in the soil was significantly reduced. Among them, under high cadmium stress, the combination of intraroot mycocystis, surface diverse mycocystis and biochar had the most significant effect.

[0048] 5. Determine the effects of the combined effects of arbuscular mycorrhizal fungi and biochar on the cadmium enrichment factor (BCF) and transfer factor (TF) of Koelreuteria paniculata.

[0049] The cadmium enrichment coefficient and transfer coefficient are used to evaluate the ability of individual plants to accumulate and transport cadmium. The calculation formulas for the transfer coefficient and enrichment coefficient are as follows:

[0050] In the formula, Cd shoot 、Cd root 、Cd soil Represent the cadmium content in the aboveground part, root system and soil respectively. Fig.11 and Fig.12 shown.

[0051] As can be seen from the figure, under low cadmium stress, the combination of intraradical Rhizospora, surface diverse cysts and biochar significantly increased the enrichment coefficient; under high cadmium stress, arbuscular mycorrhizal fungi inoculation and biochar addition, especially under the treatment of intraradical Rhizospora and biochar combination, significantly increased the root enrichment of Koelreuteria paniculata and reduced the transfer to the aboveground part, which was beneficial to reduce the cadmium content in the stems and leaves, that is, reduced the absorption and transport of cadmium in the soil.

[0052] As a potential phytoremediation species, Koelreuteria bipinnata exhibits significant heavy metal tolerance and accumulation capacity. Its well-developed root system not only increases the chance of contact with heavy metals, but also changes the chemical properties of the rhizosphere microenvironment by secreting substances such as organic acids and polysaccharides, promoting the dissolution, absorption or fixation of heavy metals. In addition, the metallothionein phytochelatin synthesized in Koelreuteria bipinnata can bind heavy metals into low-toxic complexes and store them in cell vacuoles, thereby reducing the toxic effects of heavy metals on plants.

[0053] Arbuscular mycorrhizal fungi play an important role in the remediation of heavy metal pollution. Arbuscular mycorrhizal fungi promote plant growth and increase biomass by enhancing the host plant's ability to absorb nutrients, thereby diluting the metal concentration in the tissue and reducing toxic effects. Specifically, arbuscular mycorrhizal fungi can: ① Limit cadmium transport: retain more cadmium in the root system and limit its transport to the aboveground part. ② Directly fix cadmium: enhance the accumulation and fixation of cadmium by secreting glomalin, building mycorrhizal structure, and zoning configuration, thereby reducing the toxicity of cadmium. However, the remediation effect will also be adjusted with changes in the specific plant-fungus relationship, cadmium effectiveness, and soil environmental conditions, and therefore show different remediation effects under different conditions.

[0054] As a highly effective soil conditioner, biochar significantly reduces the cadmium content in the soil through a variety of mechanisms. Its mechanisms of action include but are not limited to: ① Direct action: electrostatic attraction, ion exchange, complexation and precipitation, directly fixing cadmium ions. ② Indirect effect: indirectly affecting the biological effectiveness of cadmium by changing physical and chemical conditions such as soil matrix characteristics (such as clay minerals, organic matter), pH value and cation exchange capacity. ③ Complex characteristics: The porous structure and large surface area of ​​biochar help to adsorb heavy metal ions, while stimulating the germination of arbuscular mycorrhizal fungal spores and hyphae branching, providing a habitat for microorganisms. However, the effect of biochar is affected by the combined influence of plant species, soil biological and chemical characteristics, and the remediation effect under different conditions may vary.

[0055] The combined application of plants, arbuscular mycorrhizal fungi and biochar can significantly improve the growth and resistance of plants in Cd-contaminated soils. Specifically, it is manifested as follows: ① Significantly reduce the cadmium content in the soil: biochar and arbuscular mycorrhizal fungi work together to significantly reduce the effectiveness and mobility of cadmium in the soil through adsorption, fixation and transformation. ② Optimize cadmium distribution: biochar and arbuscular mycorrhizal fungi work together to fix more cadmium in the root system, limit its transfer to the aboveground parts, and significantly reduce the cadmium content in the aboveground parts of plants.

[0056] The present embodiment shows that, under low cadmium pollution environment, the compound pinnate Koelreuteria paniculata planted without adding arbuscular mycorrhizal fungi and biochar, the biomass is significantly reduced, the cadmium content in the plant is also significantly increased, and its enrichment coefficient (BCF) is greater than 1 under low cadmium stress, indicating that the plant has a certain phytoremediation ability under low cadmium stress; its BCF value is less than 1 under high cadmium stress, indicating that the plant not only grows impaired but also its phytoremediation ability is weakened under high cadmium stress. After adding arbuscular mycorrhizal fungi and biochar, under low cadmium stress, the combination of root endocystis and biochar can significantly reduce the cadmium content of the aboveground part of the plant, while reducing the total cadmium and DTPA active cadmium content of the soil; under high cadmium stress, the combination of root endocystis, surface diverse cystis and biochar not only significantly improves the biomass of compound pinnate Koelreuteria paniculata, reduces the effective cadmium content of the soil, reduces the cadmium content of the aboveground part of the plant, but also can significantly improve the BCF value (BCF> 1), thereby significantly improving the cadmium enrichment ability of the plant. The underlying mechanism may be that, under cadmium stress, on the one hand, the loose and porous properties of biochar applied to the soil improve the soil aeration conditions, provide a good environment for soil microorganisms, and synergistically promote the growth of arbuscular mycorrhizal fungi; on the other hand, certain nutrients will be concentrated and enriched during the pyrolysis process of preparing biochar, making the nutrient content in the biochar higher than that in the original matrix soil, which will increase the absorption and utilization of nutrients by arbuscular mycorrhizal fungi, thereby promoting plant growth and development.

[0057] Compared with the traditional restoration method of using plants, microorganisms or biochar alone, the present invention uses the combined restoration of microorganisms, plants and biochar to form a symbiotic system by co-culturing plants with microorganisms and biochar. This not only significantly reduces the cadmium content in the soil and enhances the restoration ability of the plants, but also significantly improves the growth of plants. This strategy breaks through the limitation of the existing technology that only focuses on improving plant resistance, and focuses more on the dual effects of improving plant restoration ability and growth-promoting effects, thereby maximizing the effect of plant restoration.

Claims

1. A method for reducing soil cadmium content, characterized in that: The method comprises the following steps: planting cadmium-tolerant plants in soil contaminated with cadmium, and adding arbuscular mycorrhizal fungi and biochar to the soil contaminated with cadmium.

2. A method according to claim 1, characterized in that: The cadmium-tolerant plant is Koelreuteria bipinnata.

3. A method according to claim 1, characterized in that: The arbuscular mycorrhizal fungi are ground-diffused mycocysts and / or root-diffused mycocysts.

4. The method according to claim 1, characterized in that The mass ratio of the added amount of the arbuscular mycorrhizal fungi microbial agent to the soil matrix is ​​1:

45.

5. The method according to claim 1, characterized in that The biochar is prepared by pyrolyzing rice husk, an agricultural waste, at 550° C. for 2 hours under anoxic conditions.

6. The method according to claim 1, characterized in that The amount of biochar added is 3% of the soil matrix mass.

7. The arbuscular mycorrhizal fungus according to claim 3, characterized in that For the soil with a cadmium content of 15 mg / kg, the arbuscular mycorrhizal fungus was Rhizocystis endoradiata.

8. The arbuscular mycorrhizal fungus according to claim 3, characterized in that For the soil with a cadmium content of 150 mg / kg, the arbuscular mycorrhizal fungi were Rhizocystis endoradiata and Diversicystis apophtheridia.

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