Preparation for alleviating crop plasticizer stress and its application

Abstract

The present invention discloses a formulation for alleviating plasticizer stress in crops and its application, belonging to the technical field of safe agricultural product production. This invention, for the first time, reveals the beneficial effects of reduced glutathione in alleviating plasticizer stress on crops. The use of reduced glutathione can significantly reduce the accumulation of plasticizers in crops, alleviating the inhibitory effects of plasticizers on crop growth. Furthermore, it proposes the application of reduced glutathione in crop production to effectively control and reduce plasticizer contamination of crops and avoid the adverse effects of plasticizers on the quality and safety of agricultural products.

Description

Technical Field

[0001] The present invention relates to the technical field of safe production of agricultural products, and in particular to a preparation for alleviating crop plasticizer stress and an application thereof. Background Art

[0002] Agricultural film is a vital resource for agricultural production. Mulching with film has the potential to increase warmth and conserve moisture, significantly improving agricultural production efficiency and leading to its widespread use in agricultural production. However, this has also led to a series of pollution problems. Because plastic film is not easily degradable, years of use can lead to residues and accumulation in the soil. Phthalates (PAEs), the primary plasticizer in agricultural film, are continuously released into the soil during its use cycle and after disposal, causing degradation of soil physical and chemical functions.

[0003] Phthalates, esterified derivatives of phthalic acid, are important plasticizers and softeners with strong hydrophobicity and resistance to degradation. They are widely used in agricultural production as fertilizer and pesticide carriers, making them a global soil organic pollutant (Liu et al., 2014). PAEs not only directly affect soil quality but, more seriously, are easily absorbed and accumulated in agricultural products. Exceeding certain limits can affect crop yield and quality and even harm human health through the food chain. Currently, 20 PAEs have been detected in nature, six of which, including dimethyl phthalate (DMP) and dibutyl phthalate (DBP), have established soil pollution standards (Keith et al., 1979). Furthermore, studies have shown that PAEs can cause endocrine disorders, male infertility, and have carcinogenic and teratogenic properties (Zhang et al., 2006; Wu et al., 2016). Therefore, addressing soil PAE contamination is crucial for maintaining sustainable agricultural development and public health.

[0004] my country is the world's largest tobacco producer and consumer. In recent years, the widespread use of mulching techniques in tobacco cultivation has led to an increasing amount of mulch residue in the soil, releasing large amounts of PAEs. Studies have shown that DMP and DBP have a strong inhibitory effect on tobacco growth. Therefore, there is an urgent need to develop a formulation that can reduce the absorption and accumulation of plasticizers in crops to address plasticizer stress.

[0005] Reduced glutathione (GSH) is a small, free thiol tripeptide widely found in plants and animals. It plays a vital role in numerous life processes, including maintaining protein thiol levels, thiol-disulfide bond exchange, scavenging hydroperoxides and free radicals, and transmembrane transport of amino acids (Mendoza-Cózatl et al., 2005). GSH's unique redox and nucleophilic properties enable it to participate in defense against a variety of toxic reactions, including xenobiotics, oxygen free radicals, salt damage, acid damage, and heavy metals (Ferguson and Booth, 1998; Wu et al., 2003). As an essential metabolic and regulatory substance in plants, GSH plays a crucial role in cellular defense against abiotic stresses. GSH also participates in the regulation of gene expression, cell cycle, growth, development, and protein activity, and protects plant cells against heat and chilling damage (Szalai et al., 2009). However, studies on the role of GSH in mitigating the toxicity of PAEs in tobacco and reducing their content have been limited. Summary of the Invention

[0006] The purpose of the present invention is to provide a preparation that can effectively alleviate the stress of crops to plasticizers, and apply it to crop production in a plasticizer-contaminated soil environment to reduce the accumulation of plasticizers in crops, thereby solving the adverse effects of plasticizers in the soil on the quality and safety of agricultural products.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides the use of reduced glutathione in alleviating crop plasticizer stress. Reduced glutathione can significantly alleviate the inhibitory effect of plasticizers on crop growth.

[0009] Furthermore, the plasticizer is a phthalate, which may be but is not limited to dimethyl phthalate or dibutyl phthalate.

[0010] Furthermore, the manifestations of plasticizer stress include plasticizer accumulation in crops and crop growth inhibition. Studies have shown that when crop seedlings are cultured under plasticizer stress conditions, plasticizers accumulate in the plants, significantly inhibiting crop growth, specifically manifested by a significant decrease in biomass and chlorophyll content.

[0011] Furthermore, reduced glutathione reduces the accumulation of plasticizers in crops, alleviating the inhibitory effects of plasticizers on crop growth. Studies have shown that compared with plasticizer treatment alone, the use of reduced glutathione significantly reduces plasticizer levels in plants, significantly increases biomass and chlorophyll content, and alleviates the inhibitory effects of plasticizers.

[0012] Furthermore, the crop can be but not limited to tobacco or wheat. In the present invention, the use of reduced glutathione can significantly reduce the accumulation of plasticizers in crops such as tobacco and wheat under plasticizer stress conditions.

[0013] Furthermore, the application includes applying reduced glutathione to the roots of crops during the seedling stage. When crops are grown in soil contaminated with plasticizers (dimethyl phthalate or dibutyl phthalate), applying reduced glutathione during the crop growth process can significantly alleviate the stress caused by the plasticizers that inhibit crop growth.

[0014] Furthermore, the effective concentration of the reduced glutathione is 50-100 μM.

[0015] The present invention also provides a preparation for reducing the accumulation of plasticizers in crops, comprising an active ingredient, reduced glutathione, and auxiliary ingredients required for pesticide formulations.

[0016] When the preparation is used for crop production on plasticizer-contaminated soil, its active ingredient, reduced glutathione, alleviates the inhibitory effect of plasticizers on crops by reducing the accumulation of plasticizers in crops.

[0017] Furthermore, the auxiliary component is one or more of a solvent, a cosolvent, an emulsifier, a wetting agent, a dispersant, a disintegrant, a thickener, an antifreeze agent, a preservative, a stabilizer, a film former, a defoaming agent, a colorant, a filler penetrant, and a pH regulator.

[0018] The preparation can be processed into any pesticide dosage form such as dust, wettable powder, dispersible granules, suspension, microemulsion, emulsifiable concentrate, soluble concentrate, paste, etc.

[0019] The present invention has the following beneficial effects:

[0020] This invention reveals for the first time the beneficial effects of reduced glutathione in alleviating the stress of plasticizers on crops. The use of reduced glutathione can significantly reduce the accumulation of plasticizers in crops and alleviate the inhibitory effect of plasticizers on crop growth. It further proposes the application of reduced glutathione in crop production to effectively control and reduce plasticizer contamination of crops and avoid the adverse effects of plasticizers on the quality and safety of agricultural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effects of DMP and GSH treatments on the chlorophyll content of tobacco seedlings. a, b, and c indicate significant differences at the 0.05 level.

[0022] Figure 2 Phenotypic differences of tobacco seedlings under DBP and GSH treatments.

[0023] Figure 3 The effects of DBP and GSH treatments on the chlorophyll content of tobacco seedlings. a and b indicate significant differences at the 0.05 level.

[0024] Figure 4 The effect of 50 μM GSH treatment on the growth of wheat seedlings under DBP stress.

[0025] Figure 5 Effects of 100 μM GSH treatment on the growth of wheat seedlings under DBP stress. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with specific examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all fall within the scope of the present invention.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0028] Reduced glutathione (GSH), CAS: 70-18-8; dimethyl phthalate (DMP), CAS: 131-11-3; dibutyl phthalate (DBP), CAS: 84-74-2.

[0029] 1 / 5 Hogland nutrient solution (mg L -1 ): K2SO4 130.7, MgSO4 . 7H2O 160.2, KCl7.5, KH2PO413.6, Ca(NO3)2 . 4H2O 472.3, Fe-EDTA-Na 36.7, MnSO4 . H2O0.006, ZnSO4 . 7H2O 0.287, CuSO4 . 5H2O 0.025, (NH4)6Mo7O 24 . 4H2O 0.006, H3BO3 0.062.

[0030] Example 1: GSH alleviates tobacco DMP stress

[0031] 1. Experimental methods

[0032] The tobacco variety used in this example was CX26. Tobacco seeds were sterilized and germinated in the dark. After germination, they were transplanted to a vermiculite culture medium. Tobacco seedlings were cultured in a constant-temperature, light-controlled incubator at 22°C / 18°C. Healthy, uniformly growing tobacco seedlings were selected at the two-leaf, one-heart stage and transplanted to a 1 / 5 Hogland nutrient solution. The pH was adjusted to 5.8 ± 0.1 using HCl and NaOH.

[0033] After 7 days of pre-culture, the cells were treated with three treatments: (1) control: BNS (basic culture medium, 1 / 5 Hogland nutrient solution); (2) DMP: BNS + 50 mg L -1 DMP; (3) DMP + GSH: BNS + 50mg L -1 DMP+100μM GSH. In the DMP group, DMP was first dissolved in acetone and then added to the BNS culture medium to prepare a final concentration of 50 mg L -1 The final concentration of DMP in the DMP+GSH group was 50 mg L -1 , the final concentration of GSH was 100 μM.

[0034] Sampling was performed 7 days after treatment, and the tobacco plants were divided into aboveground and underground parts for biomass measurement (Lin et al., 2022) and DMP content determination; chlorophyll content was measured using a handheld SPAD instrument.

[0035] The DMP extraction and determination method is as follows: Prepare a DMP standard solution; weigh 0.5 g of ground sample and place it in a 25 mL stoppered Erlenmeyer flask. Add 2 mL of dichloromethane:acetone (3:1, v / v) and 50 μL of internal standard solution. Ultrasonic extraction is performed for 40 minutes. An appropriate amount of the extract is placed in a centrifuge tube, solid dispersant is added, and centrifugation is performed for 10 minutes at 4000 rpm. The supernatant is collected for GC-MS analysis.

[0036] 2. Experimental results

[0037] 2.1 Chlorophyll determination

[0038] like Figure 1 As shown in the figure, the chlorophyll content was significantly increased by 15.5% after the addition of GSH compared with the DMP treatment alone.

[0039] 2.2 Biomass determination

[0040] As shown in Table 1, compared with the DMP treatment alone, the dry weights of the aboveground and underground parts increased significantly by 42.2% and 60.0%, respectively, after the addition of GSH.

[0041] Table 1. Dry weight of aboveground and underground parts of tobacco under DMP and GSH treatments (g plant-1 )

[0042]

[0043] Note: a, b indicate significant differences at the 0.05 level.

[0044] 2.3DMP content determination

[0045] As shown in Table 2, compared with the DMP treatment alone, the DMP content in the underground part decreased significantly by 49.0% after the addition of GSH.

[0046] Table 2. DMP content in aboveground and underground parts of tobacco under DMP and GSH treatments (mg kg -1 )

[0047]

[0048] Note: a, b and c indicate significant differences at the 0.05 level.

[0049] The above results showed that DMP significantly inhibited tobacco growth, and the inhibitory effect of DMP was significantly alleviated after the addition of GSH.

[0050] Example 2: GSH alleviates tobacco DBP stress

[0051] 1. Experimental methods

[0052] The tobacco varieties and germination methods used in this example were the same as those in Example 1. Treatments were performed after 7 days of pre-culture, with three treatments: (1) Control: BNS (basic culture medium); (2) DBP: BNS + 50 mg L -1 DBP; (3) DBP + GSH: BNS + 50mgL -1 DBP+100μM GSH. In the DBP group, DBP was first dissolved in acetone and then added to the BNS culture medium to prepare a final concentration of 50mg L -1 The final concentration of DBP in the DBP+GSH group was 50 mg L -1 , the final concentration of GSH was 100 μM.

[0053] Samples were taken 7 days after treatment, and the tobacco plants were divided into aboveground and underground parts for determination of DMP content and biomass; and chlorophyll content was determined.

[0054] 2. Experimental results

[0055] 2.1 Tobacco seedling phenotype

[0056] like Figure 2 As shown in the figure, DBP significantly inhibited tobacco growth, and the inhibitory effect of DBP was significantly alleviated after the addition of GSH.

[0057] 2.2 Chlorophyll determination

[0058] like Figure 3 As shown in Figure 3, the chlorophyll content increased by 7.6% after the addition of GSH compared with the DBP treatment alone.

[0059] 2.3 Biomass determination

[0060] As shown in Table 3, compared with the DBP treatment alone, the dry weights of the aboveground and underground parts increased significantly by 51.1% and 90.1%, respectively, after the addition of GSH.

[0061] Table 3. Dry weight of aboveground and underground parts of tobacco under DBP and GSH treatments (g plant -1 )

[0062]

[0063] Note: a, b and c indicate significant differences at the 0.05 level.

[0064] 2.4DBP content determination

[0065] As shown in Table 4, the DMP contents in the aboveground and underground parts of the DBP+GSH treatment decreased significantly compared with the DBP treatment.

[0066] Table 4. DBP content in aboveground and underground parts of tobacco under DBP and GSH treatments (mg kg -1 )

[0067]

[0068] Note: a, b and c indicate significant differences at the 0.05 level.

[0069] Example 3: GSH alleviates wheat DBP stress

[0070] 1. Experimental methods

[0071] The wheat variety used in this example is Yangmai 20. Seeds with relatively consistent grain size and plumpness were selected, disinfected with 3% hydrogen peroxide for 20 minutes, rinsed thoroughly with tap water, and sown on moist sand. After 7 days (at the one-leaf, one-heart stage), uniformly growing seedlings were selected and transplanted into a black hydroponic box.

[0072] The seedlings were cultured in 1 / 5 Hoagland nutrient solution until they had two leaves and one heart, and then treated. Four treatments were set up: (1) Control: BNS (basic culture medium); (2) DBP: BNS + 50 mg L -1 DBP; (3) DBP+GSH (50μM): BNS+50mg L - 1DBP+50μM GSH; (4)DBP+GSH(100μM), BNS+50mg L -1 DBP+100μM GSH. In the DBP group, DBP was first dissolved in acetone and then added to the BNS culture medium to prepare a final concentration of 50mg L -1 The final concentration of DBP in the DBP+GSH (50 μM) group was 50 mg L -1 The final concentration of GSH was 50 μM; the final concentration of DBP in the DBP+GSH (100 μM) group was 50 mg L -1 , the final concentration of GSH was 100 μM.

[0073] Samples were taken 7 days after treatment, and the wheat plants were divided into aboveground and underground parts for determination of DBP content and biomass.

[0074] 2. Experimental results

[0075] 2.1 Wheat seedling phenotype

[0076] like Figure 4 and Figure 5 As shown in the figure, DBP stress significantly inhibited wheat growth, and the inhibitory effect of DBP was significantly alleviated after adding 50μM and 100μM GSH.

[0077] 2.2 Biomass determination

[0078] As shown in Tables 5 and 6, compared with the control, the dry weight of the aboveground and underground parts of wheat treated with DBP significantly decreased by 25.9% and 24.4%, respectively. After adding 50 μM GSH, the dry weight of the aboveground and underground parts significantly increased by 32.9% and 43.3%, respectively. After adding 100 μM GSH, the dry weight of the aboveground and underground parts significantly increased by 22.0% and 14.9%, respectively.

[0079] Table 5. Effects of 50 μM GSH on the growth of wheat seedlings under DBP stress (g plant -1 )

[0080]

[0081] Note: a, b indicate significant differences at the 0.05 level.

[0082] Table 6. Effects of 100 μM GSH on the growth of wheat seedlings under DBP stress (g plant -1 )

[0083]

[0084] Note: a, b indicate significant differences at the 0.05 level.

[0085] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. The application of reduced glutathione in alleviating crop plasticizer stress, characterized in that: The plasticizer is dimethyl phthalate or dibutyl phthalate; and the crop is tobacco or wheat.

2. The use according to claim 1, characterized in that The manifestations of plasticizer stress include accumulation of plasticizers in crops and inhibition of crop growth.

3. The use according to claim 1, characterized in that The reduced glutathione reduces the accumulation of plasticizers in crops and alleviates the inhibitory effect of plasticizers on crop growth.

4. The use according to claim 1, wherein The application comprises: applying reduced glutathione to the roots of crops during the seedling stage.

5. The use according to claim 1, characterized in that The effective concentration of the reduced glutathione is 50-100 μM.

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