Method for intercropping plants containing rhizobium or ectomycorrhiza to restrain PAEs pollutants in tea garden soil

Abstract

The invention provides a method for intercropping plants containing rhizobium or ectomycorrhiza to restrain PAEs pollutants in tea garden soil, and belongs to the field of tea science and the field of PAEs pollution treatment. The method aims at solving the problem that tea garden soil is polluted by PAEs pollutants, and the tea quality is affected. According to the method, plants are planted in the PAEs contaminated tea garden, the plants containing rhizobium are soybeans, flemingia macrophylla and amorpha fruticosa, and chestnut trees grow ectomycorrhiza. The plant rhizosphere can conserve microorganisms, the root system can generate secretions, and the microorganisms and the secretions can decompose PAEs. The chestnut trees are preferably intercropped, and the good effect of inhibiting PAEs pollutants is achieved under the planting density of (9-11) m * (9-11) m; the planting density does not affect photosynthesis of the tea trees, manual tea picking, pruning, fertilization, weeding and ploughing in the field, the content of PAEs can be effectively reduced, movement of the PAEs in the tea garden is prevented, increase of the content of the PAEs is restrained, and technical reference is provided for quality control and safety of the tea leaves.

Description

Technical Field

[0001] The invention belongs to the field of tea science and PAEs pollution control, and specifically relates to a method for inhibiting PAEs pollutants in tea garden soil by intercropping plants containing rhizobia or ectomycorrhizae. Background Art

[0002] PAEs, or phthalic acid esters, are plastic additives that enhance flexibility, or plasticizers. Currently, their usage accounts for 80% of the total plasticizers. Due to their teratogenic, carcinogenic, and mutagenic potential, many countries and regions stipulate that the maximum amount of PAEs added to plastic products should not exceed 30%.

[0003] PAEs have become a class of international environmental pollutants, including more than 30 homologues. They pollute soil, water and air. Studies have shown that tea tree roots can absorb PAEs in soil and solution. If the soil is seriously polluted by PAEs, PAEs are likely to enter the tree through the roots and be transported to the leaves. Fresh tea leaves are picked and processed into commodities, and PAEs will enter the human body when tea drinkers brew and drink tea.

[0004] PAEs in tea garden soil are one of the new sources of pollution for fresh tea leaves, and it is critical to prevent and inhibit soil PAEs from entering the root system of tea trees. Chestnut trees contain ectomycorrhizae, while soybeans, Amorpha fruticosa, and Echinops chinensis contain rhizobia. Ectomycorrhizae and rhizobia nourish rhizosphere microorganisms, and the root system also produces secretions, in which the microorganisms and secretions can decompose PAEs. The present invention intends to screen out plant species that inhibit PAEs and their suitable planting patterns from plants such as chestnut trees, soybeans, Amorpha fruticosa, and Echinops chinensis through comparative experiments. Summary of the invention

[0005] In order to solve the pollution problem of PAEs in tea garden soil, the present invention provides a method for inhibiting PAEs pollutants in tea garden soil by intercropping plants containing rhizobia or ectomycorrhizae, which utilizes the root-nourishing microorganisms and root secretions of the intercropped plants containing rhizobia ectomycorrhizae in the tea garden to decompose PAEs, thereby preventing and inhibiting the movement of PAEs in the tea garden soil and the growth of their content, thereby inhibiting and reducing the entry of PAEs into tea leaves from the roots.

[0006] The present invention provides a method for inhibiting PAEs pollutants in tea garden soil by intercropping rhizobia or ectomycorrhizal plants, which is achieved by the following steps:

[0007] In tea gardens contaminated with PAEs, plants containing rhizobia or ectomycorrhizae are intercropped, and the root systems or rhizosphere microorganisms of the plants containing rhizobia or ectomycorrhizae are used to absorb and decompose PAEs pollutants; the plants containing rhizobia are soybeans, broadleaf schizonepeta or Amorpha fruticosa, and the plants containing ectomycorrhizae are chestnut trees.

[0008] Preferably, the plants containing rhizobia or ectomycorrhizae are evenly planted at equal intervals in the horizontal and vertical directions in the tea garden.

[0009] Preferably, the plant containing rhizobia is soybean, and the plant containing ectomycorrhiza is chestnut.

[0010] Among them, in tea gardens with less pollution, for example, when the total content of PAEs in the soil of the tea garden is less than 300μg / kg, soybeans, broad-leaved Amorpha or Sophora japonica are planted, and soybeans are preferred; in tea gardens with more serious pollution, for example, when the total content of PAEs in the soil of the tea garden is greater than 300μg / kg, chestnut trees are preferably planted.

[0011] Preferably, when chestnut trees are intercropped, the planting distance between chestnut trees is 9m to 11m, and chestnut trees are planted in a plane array with equal spacing in the tea garden. Preferably, the planting distance between chestnut trees is 10m, that is, they are planted in the tea garden at a planting density of 10m×10m.

[0012] Preferably, the shading degree or crown coverage of the chestnut tree crown does not exceed 1 / 3 of the tea garden area, and the crown diameter is ≤3.3m.

[0013] Compared with the prior art, the method of the present invention inhibits PAEs pollutants in tea garden soil by intercropping plants containing rhizobia or ectomycorrhizae. The method does not use chemical reagents and is pollution-free to the environment and the tea garden. Intercropping chestnut trees does not affect the normal operation of the tea garden. The present invention reasonably controls the planting density of chestnut trees, so that the effect on tea yield is small, and the PAEs yield in the tea garden can be effectively reduced.

[0014] For tea gardens with heavy pollution, the method of planting chestnut trees is preferred. This method is continuously effective. As the chestnut trees grow, the ability of the roots to absorb and decompose PAEs pollutants is expected to be further enhanced. The method of the present invention is convenient for subsequent maintenance, and only requires regular pruning of the chestnut tree crowns to reduce their impact on the number of tea leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a graph showing the changes in total PAEs content in pure tea gardens, soybean-tea intercropping, large-leaf ganjin-tea intercropping and Amorpha paniculata-tea intercropping in different seasons. DETAILED DESCRIPTION

[0016] Example 1 Preliminary test

[0017] 1 Experimental design

[0018] 1.1 Sample collection

[0019] In January 2021, a large tea garden with a relatively light PAEs pollution level of the Golden Bud variety was selected in the tea area of ​​Songyang County, Zhejiang Province. The tea trees were 4 years old, 80-90 cm tall, 90-100 cm wide, planted in double rows with a row spacing of 1.5 m. Another large tea garden with a relatively heavy PAEs pollution level was selected in the tea area of ​​Songyang County. The tea trees were planted in double rows with the same variety, tree age, tree height, tree width, row spacing, etc. as those in the tea garden with relatively light PAEs pollution.

[0020] On January 2, 2021, the five-point sampling method was used to select sampling points in these two tea gardens.

[0021] Next, remove fallen leaves and other impurities from the soil surface to expose the topsoil. Then use a 20 cm soil drill to collect soil from 0-10 cm and 10-20 cm, i.e., the upper and lower surface layers, respectively, and mix the five points to form a soil sample. These soil samples were placed in brown glass bottles with polytetrafluoroethylene caps and stored at 4°C awaiting analysis. The soil samples were freeze-dried to remove moisture, then ground and passed through a 60-mesh metal sieve (0.25 mm) for extraction.

[0022] On the sampling day of January 2, the sampling was repeated 5 times. The 5 samples were analyzed separately and the average was calculated.

[0023] 1.2 Sample extraction

[0024] To reduce background contamination, all glassware and polytetrafluoroethylene centrifuge tubes were soaked in potassium dichromate solution before the experiment, then washed with pure water and dried. Plastic containers were avoided throughout the experiment, and contact with plastic items was minimized. To monitor background contamination, each batch of samples was tested for solvent blank and instrument blank, and a procedure blank was tested every 12 samples.

[0025] For each soil sample, PAEs were extracted using the following steps:

[0026] Ⅰ. Mix 5 g of soil sample and 10 g of anhydrous sodium sulfate in a 50 mL polytetrafluoroethylene test tube. After ensuring that the mixture is evenly mixed, add 20 mL of ethyl acetate and shake to further mix;

[0027] Ⅱ. After 30 min of ultrasonic treatment, the samples were centrifuged at 4 °C and 3000 rpm for 3 min, and all the supernatants were collected;

[0028] III. Add 10 mL of ethyl acetate to the sample and perform ultrasonic treatment again for 10 min, then centrifuge at 4°C, 3000 rpm for 3 min. Collect the supernatant and repeat step II twice;

[0029] IV. Collect the supernatant and repeat step III twice. Evaporate all the supernatants to near dryness using a reduced pressure rotary evaporator at 33°C and 50 rpm, and then add 3 mL of ethyl acetate to re-dissolve.

[0030] V. Use 6 mL of ethyl acetate to activate a solid phase extraction column (Dikma AL-B-1000 mg / 6 mL Glass), discard the first outflowing liquid, then add the liquid to be purified into the column, and collect all the outflowing liquid.

[0031] Ⅵ. Filter all collected effluent through a 0.22 μm nylon filter membrane, and then concentrate to 0.5 mL using nitrogen purge method and wait for instrument analysis.

[0032] 1.3 GC-MS analysis conditions

[0033] The quantitative analysis of PAEs uses an Agilent 6890GC-5975B MSD system. The chromatographic column used in this embodiment is an HP-5MS quartz capillary column (30m×250μm×0.25μm). The GC column box temperature is maintained at 60°C for 1 min, heated to 220°C at 20°C / min and maintained for 1 min, then heated to 290°C at 5°C / min and maintained for 5 min. 1μL of sample enters the GC-MS in splitless mode with an injection temperature of 280°C. The carrier gas is helium with a concentration of 99.999%. The ion source temperature is 230°C and the ion energy is 70eV. The analysis is performed using the ion detection mode (SIM). The recoveries of the seven PAEs in the spiked matrix blank samples are (79.05-113.51%), and the relative standard deviations are (4.07-15.54%). The detection limit (LOD) ranged from (0.701 to 6.088 μg / kg) and the quantification limit (LOQ) ranged from (2.336 to 20.293 μg / kg) calculated by three times signal-to-noise ratio and ten times signal-to-noise ratio. When the test data was lower than the LOD value, the 1 / 2 LOD value was used for statistical analysis.

[0034] 1.4 Data Analysis

[0035] R-4.2.3 was used for data visualization and SPSS-26.0 was used for statistical tests.

[0036] 2 Test results

[0037] The total amounts of PAEs in tea gardens with lighter PAEs pollution and tea gardens with heavier PAEs pollution were 171.1μg / kg and 561μg / kg respectively.

[0038] Example 2 PAEs content difference between soybean-tea, large-leaf gentian-tea, Amorpha fruticosa-tea and pure tea garden (CK tea garden) in tea gardens with lower PAEs pollution

[0039] 1 Experimental design

[0040] 1.1 Arrangement of four plots of the Golden Bud tea garden with a relatively low PAEs pollution level

[0041] In the tea garden with a relatively light PAEs pollution degree in the "Preliminary Test of Example 1", four plots of 2 mu each were selected as soybean-tea intercropping tea garden, large-leaf spatholobus-tea intercropping tea garden, Amorpha fruticosa-tea intercropping tea garden, and a pure tea garden without intercropping. The four plots were arranged side by side with a spacing of 20m.

[0042] From 2021 to 2024, on April 10, June 10 and August 10 every year, soybeans will be planted in the designated soybean-tea intercropping area with a density of 5m×5m, with 2 soybean seedlings left in each pocket, and the soybean roots, branches and leaves will remain in the tea garden.

[0043] On April 10, 2021, we planted large-leaf mulberry in the designated large-leaf mulberry-tea intercropping area, with a density of 5m×5m, and planted 2 seedlings in each pocket. If dead seedlings are found before the end of 2024, they will be replanted immediately.

[0044] On April 10, 2021, Amorpha fruticosa was planted in the designated intercropping area between Amorpha fruticosa and tea, with a density of 5m

[0045] ×5m, plant 2 seedlings in each bag. If there are dead seedlings before the end of 2024, replant them immediately.

[0046] 1.2 Sample collection, sample extraction and analysis

[0047] On January 2, 2025, according to the sampling method of "Example 1 Preliminary Test", sample collection, sample extraction, and instrumental analysis were carried out in soybean-tea, large-leaf Amorpha-tea, Amorpha-tea, and pure tea garden (CK tea garden) according to the five-point sampling method.

[0048] 2 Test results

[0049] Sample analysis results on January 2, 2025: the total content of PAEs in soybean-tea intercropping tea garden, the total content of PAEs in large-leaf Aconite-tea intercropping tea garden, the total content of PAEs in Amorpha fruticosa-tea intercropping tea garden, and the total content of PAEs in pure tea garden were 170.13±19.00μg / kg, 178.01±14.64μg / kg, 185.91±15.14μg / kg and 211.11±27.55μg / kg, respectively.

[0050] The results showed that the differences in the total PAEs content between the first three intercropping tea gardens and the pure tea garden (CK tea garden) were all significant (P < 0.05). The differences in the total PAEs content between the first three intercropping tea gardens were not significant. The total PAEs content in the soybean-tea intercropping tea garden was slightly lower.

[0051] Example 3 Seasonal changes in PAEs content in soybean-tea, large-leaf gentian-tea, Amorpha fruticosa-tea and pure tea gardens with relatively light PAEs pollution

[0052] 1 Experimental design

[0053] In the four plots of Example 2 above, in the summer (June 2), autumn (September 2), winter (December 2) of 2022, and spring (March 2) of 2023, samples were collected, extracted and analyzed in the same manner as in the "Preliminary Test of Example 1".

[0054] 2 Test results

[0055] The test results of the first anniversary showed that in the tea gardens with lower PAEs pollution, the soil PAEs content in the tea gardens with three crops intercropped with tea and the CK tea gardens fluctuated seasonally, but the soil PAEs content in the intercropped tea gardens was lower than that in the CK tea gardens, and the total PAEs content in the soybean-tea intercropped tea gardens was slightly lower than that in the other three types of tea gardens ( Figure 1 ), indicating that intercropping rhizobia crops can effectively inhibit PAEs in tea garden soil, and the inhibitory efficacy of different intercrops is different.

[0056] Example 4 PAEs content difference between chestnut-tea intercropping tea garden and pure tea garden (CK) in tea garden with high PAEs pollution level

[0057] 1 Experimental design

[0058] In the tea garden with a heavy pollution degree of PAEs in the "Preliminary Test of Example 1", three plots of 2 mu each were selected in this tea garden on April 10, 2021, which were used as chestnut-tea intercropping tea garden (intercropping distance 10m×10m), pure tea garden (CK tea garden), chestnut-tea intercropping tea garden (intercropping distance 20m×20m). The plots are arranged side by side with a spacing of 20m. The chestnut tree is 2.5m tall and has a breast diameter of 3cm. As the chestnut tree grows, the crown gradually expands. Before sampling on January 2, 2025, the crown is pruned to keep it ≤3.3m.

[0059] Sampling was conducted on January 2, 2025, and PAEs sample collection, sample extraction and analysis were the same as in “Preliminary Test in Example 1”.

[0060] 2 Test results

[0061] Analysis results on January 2, 2025: The total PAEs levels in the chestnut-tea intercropping tea garden (intercropping distance 10m×10m), pure tea garden, and chestnut-tea intercropping tea garden (intercropping distance 20m×20m) were 592.01±65.41μg / kg, 780.11±95.88μg / kg, and 725.12±91.76μg / kg, respectively.

[0062] The t-test showed that there was a significant difference in the total amount of PAEs between chestnut-tea intercropping (intercropping distance 10m×10m) and that of pure tea gardens; there was no significant difference in the total amount of PAEs between pure tea gardens and chestnut-tea intercropping (intercropping distance 20m×20m).

[0063] Therefore, it is believed that chestnut trees have a strong ectomycorrhizal root system, which can produce fungi or secrete enzymes to degrade PAEs. The intercropping of chestnut trees and tea at a density of 10m×10m has a good inhibitory effect on PAEs.

Claims

1. A method for inhibiting PAEs pollutants in tea garden soil by intercropping plants containing rhizobia or ectomycorrhizae, characterized in that: This is accomplished by following these steps: In tea gardens with PAEs pollution, plants containing rhizobia or ectomycorrhizae are intercropped, and microorganisms conserved in the root system or rhizosphere are used to absorb and decompose PAEs pollutants; the plants containing rhizobia are soybeans, broadleaf spatholobus, and Amorpha fruticosa, and the ectomycorrhizal plants are chestnut trees.

2. The method according to claim 1, characterized in that The plants containing rhizobia or ectomycorrhizae are evenly planted in the tea garden at equal intervals in the horizontal and vertical directions.

3. The method according to claim 1, characterized in that The rhizobium-containing plant is soybean, and the ectomycorrhizal plant is chestnut.

4. The method according to claim 1, characterized in that: When intercropping chestnut trees, the planting distance between chestnut trees is 9m to 11m, and chestnut trees are planted at equal spacing in the horizontal and vertical directions in the tea garden.

5. The method according to claim 4, characterized in that The planting distance between chestnut trees is 10m, the shading degree or crown coverage of the chestnut tree crown shall not exceed 1 / 3 of the tea garden area, and the crown diameter shall be ≤3.3m.

6. The method according to claim 1, characterized in that When the total content of PAEs in the soil of the tea garden is less than 300μg / kg, soybean, broadleaf clematis or Amorpha fruticosa should be intercropped; when the total content of PAEs in the soil of the tea garden is greater than 300μg / kg, chestnut trees should be intercropped.

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