Dissolved Organic Matter from Earthworm Casts Restrained the Phytotoxicity of Soil Glyphosate to Citrus (Poncirus trifoliata (L.) Raf.) Plants

Abstract

A large amount of glyphosate enters the soil at a high frequency, forming “pseudo-persistent” pollutants that, in turn, threaten soil ecological function and crop growth. Earthworm casts (EWCs) are a sound organic alternative to chemical fertilizers to promote crop growth. Dissolved organic matter from EWCs (EWC-DOM) is supposed to be a more mobile and bioavailable fraction. However, the effect of EWC-DOM on the phytotoxicity of glyphosate remains largely unknown. This study examines glyphosate-induced oxidative stress and its impact on antioxidant and detoxification enzymes in citrus plants grown in soils with and/or without EWC-DOM. The results suggest that EWC-DOM could reduce the membrane lipid peroxidation level, thus slowing down the aging of plants in order to maintain stronger resilience, with more active antioxidant enzymes (including SOD, POD, and CAT) and detoxification enzymes (including GST, laccase, CPR, and UGTs) that could effectively remove reactive oxygen species (ROS) caused by glyphosate stress, thereby alleviating the damage of ROS accumulation on plant tissues. Our data indicate that incorporating EWC-DOM should be a sound way to protect citrus plants from the phytotoxicity caused by using glyphosate for orchard weeding. This has major implications for the sustainable and healthy development of citrus production.

1. Introduction

The excellent herbicidal performance of glyphosate and its initial reputation as an ecofriendly herbicide have contributed to its extensive global use [1]. Especially in citrus plantations, glyphosate is the most preferred herbicide [2,3]. China is one of the world’s major citrus growers, ranking first in cultivated area and total output. China has also led the world to producing and using glyphosate [4]. Citrus, the greatest-yield fruit in the world, is widely grown in tropical and subtropical regions where high-temperature and high-humidity climates are conducive to the breeding and spread of weeds. Glyphosate has frequently been applied in large quantities for orchard weeding, inevitably forming “pseudo-persistent” contamination in soil [5]. Then, residual glyphosate in soil may not only be absorbed into citrus plants via the roots, damaging citrus growth, fruit yield, and quality, but also accumulate in the food chain, causing harm to animal and human health [6,7,8,9].

In the latest decade, with the continuous development of chemical fertilizer reduction and organic substitution in China, earthworm casts (EWCs)—digested organic waste from earthworms with relatively higher microbial community, nitrogen content, and specific surface, but a lower carbon–nitrogen ratio [10,11]—which had first mainly been used in horticultural crops, have been popularized in agriculture, especially in fruit-tree (such as citrus) production [12,13]. Throughout the years, stemming from the eager public concern about the farmland soil pollution caused by pesticide application, the removal effect of earthworms on residual pesticides in soil has been extensively studied, and some studies have mentioned EWCs [14]. Solid EWCs have played a role in the degradation of soil pollutants to alleviate their bioavailability in and stress impact on crop growth [15]. DOM is promptly released to soil with the application of organic matter and is more readily absorbed by plants. It is the most active component of organic fertilizers [16]. In soil solutions, DOM can act as the main transport carrier or co-solvent of organic pollutants, thus impacting their migration, transformation, and toxicity in soil [12]. Especially glyphosate, which is a water-soluble pesticide, might be more heavily impacted by EWC-DOM than by solid EWCs in terms of its bioavailability, and its phytotoxicity would fully reflect crop plants. However, the role of dissolved organic matter (DOM) derived from EWCs (EWC-DOM) remains an open question. Given the above information, this paper (1) investigates the response of citrus seedlings to glyphosate in soil in the presence or absence of EWC-DOM and the potential regulatory capacity of EWC-DOM in this process, (2) explores competent indicators for the level of glyphosate pollution in soil–crop systems, and (3) decodes the role of enzymes in living fruit trees in response to pesticide stress in soil culture systems, hoping to inspire the formulation of strategies to mitigate the negative effects of soil glyphosate on crop growth.

2. Materials and Methods

2.1. Materials

Organic phosphine herbicide glyphosate with >99% purity was procured from Dr. Ehrenstorfer GmbH, Augsburg, Germany. Citrus seeds were purchased from the Hainan Academy of Agricultural Sciences (Danzhou, China). The same soil sample as that in our previous study was used in this study; the composition and physico-chemical properties of the blank soil are summarized in Jiang et al. [17]. Uncontaminated topsoil was collected from the Danzhou branch of the Chinese Academy of Tropical Agriculture, Hainan province (19.50° N, 109.48° E). After having been air-dried, the soil samples were sieved to <3 mm to dispose of stones, dead branches, and other debris. EWCs were procured from the local earthworm farm of the Danzhou Academy of Agricultural Sciences. They were air-dried and passed through a 2 mm sieve, mixed with ultrapure water at a solid/liquid ratio of 1:10 (W/V), shaken at 200 rpm for 16 h at 20 °C, and then centrifuged at 6000 rpm for 15 min at 4 °C. The supernatant was suction-filtered through a 0.45 μm filter membrane to obtain EWC-DOM for future use. The concentration of the EWC-DOM solution was measured with a TOC analyzer, and its concentration was characterized via water-soluble organic carbon (DOC) [18].

2.2. Plant Cultivation and Treatment

The experiment was designed with six treatments: Control, undisturbed soil; E1, soil with 40 mg DOC kg⁻¹ of EWC-DOM; E2, soil with 80 mg DOC kg⁻¹ of EWC-DOM; Gly, soil with 12.15 mg kg⁻¹ of Gly; G + E1, soil with 12.15 mg kg⁻¹ of Gly in the presence of 40 mg DOC kg⁻¹ of EWC-DOM; G + E2, soil with 12.15 mg kg⁻¹ of Gly in the presence of 80 mg DOC kg⁻¹ of EWC-DOM. The test concentration of Gly was determined to be 12.15 mg kg⁻¹ on the basis of the physiological and biochemical responses of cowpea plants to Gly stress in the soil during our preliminary experiment, and the actual application amount of glyphosate sprayed in the field and its accumulated residues in the farmland [12,19]. The concentration of EWC-DOM selected in the experiment was also comprehensively considered on the basis of its economic and practical use in farmland. Citrus seeds were sterilized in hot water at 50 °C for 50 min and then cultivated to germinate. Seeds with consistent germination were evenly sown in 1 L plastic jars containing 1000 g of different soil treatments and placed in an artificial climate box for cultivation. The humidity was adjusted to 70%, the light/dark (27/22 °C) cycle was conditioned with 300 μmol m⁻² s⁻¹ artificial illumination during a 14 h photoperiod, and water was regularly added every day via a weighing method. The shoots and roots of the plants were collected for the detection and analysis of total chlorophyll, cellular lipid peroxides, and the activities of antioxidant enzymes and detoxification enzymes after 10 weeks of incubation.

2.3. Plant Growth Parameters

The elongation of the main stem and main root of the plants were measured with a tape measure. In a constant-temperature oven, fresh plant samples were dried at 105 °C for 20 min and then at 80 °C for 48 h, until the dry mass was weighed [17].

2.4. Plant Physiological Parameters

The fresh leaves were cut into small pieces and extracted in the dark for 24 h. The total chlorophyll content in the supernatant was determined via spectrophotometer [20]. The membrane lipid peroxidation level of plants was characterized via measuring the content of the thiobarbituric acid reaction substrates (TBARS) [21].

2.5. Antioxidant Enzyme and Detoxification Enzyme Activity Assay

The fresh leaves and roots of citrus seedlings were placed in a mortar and ground into a homogenate with 50 mmol L⁻¹ sodium phosphate buffer. The homogenates were centrifuged at 12,000× g and 4 °C for 20 min to obtain the crude enzyme extract, which was used to determine the activities of antioxidant enzymes and detoxification enzymes. According to Banik et al., the Coomassie Brilliant Blue G-250 method was used to measure the soluble protein content [22]. Superoxide dismutase (SOD, EC 1.15.1.1) has the capacity to suppress the reversion of nitro-blue tetrazolium (NBT) under light. Thus, SOD activity was evaluated via photochemical reduction of NBT [23]. The reaction mixture was 3 mL of 50 mM phosphate buffer (pH 7.8) containing 10 mM methionine, 1.17 mM riboflavin, 56 mM NBT, and 30 μL enzyme extract. The absorbance of the solution was measured at 560 nm. 50% of inhibition of the photoreduction of NBT to blue was defined as one unit of SOD activity. Catalase (CAT, EC 1.11.1.6) activity was determined through observing the decline velocity in absorbance of H₂O₂ at 240 nm within 4 min according to the method of Ishibashi et al. [24]. The reaction mixture was 3 mL of 100 mM potassium phosphate buffer (pH 7.0) containing 15 mM H₂O₂, and 50 µL of enzyme extract. Peroxidase (POD, EC 1.11.1.7) can catalyze the oxidation of guaiacol. Accordingly, the POD activity was determined via guaiacol colorimetry [25]. The activity was assayed for 3 min in a reaction solution (3 mL final volume) composed of 100 mM potassium phosphate buffer (pH 7.0), 20 mM guaiacol, 10 mM H₂O₂, and 50 µL of enzyme extract. Glutathione S-transferase (GST, EC 2.5.1.18) activity was determined through calculating the growth rate of absorbance caused by the GST catalyzed GSH-CDNB conjugation at 340 nm within 3 min [26]. A total of 3 mL of the reaction mixture contained 100 mM potassium phosphate buffer (pH 7), 1 mM GSH, 1 mM CDNB, and proteins from enzyme extract. Laccase (Laccase, EC 1.10.3.2) activity was determined through measuring the change rate of the absorbance of the reaction mixture consisted of 5 mM ABTS, acetate buffer (pH 4.2) and 100 μL of enzyme extraction solution, at 420 nm within 5 min [27]. NADPH-cytochrome P450 reductase (CPR, EC 1.6.2.4) was the catalyst for the reduction of cytochrome C using NADPH. The reduced cytochrome C had a characteristic absorption peak at 550 nm. Thus, CPR activity was calculated through measuring the increase rate of absorbance of the reduced cytochrome C at 550 nm within 5 min [28,29]. Following the method described by Suzuki et al. [30], UDP-glycosyltransferases (UGTs, EC 2.4.1.17) activity was calculated through monitoring the depletion of the sugar receptors content in the reaction solution with high-performance liquid chromatography equipped with an ultraviolet detector at 317 nm and a reverse C₁₈ column (Thermo, 250 mm × 4.6 mm i.d.), using methanol:water (v:v) = 3:2 as mobile phase with the flow rate of 0.6 mL min⁻¹.

2.6. Statistical Analysis

Each treatment was repeated in triplicate, and each replicate contained at least 5 citrus seedlings. All results shown in the paper were the mean of three replications. The values in the experimental graphs were calculated using “mean ± standard deviation”. Significant differences between the results from the experimental group were determined using a one-way analysis of variance (p < 0.05). Statistical analysis was conducted with the program SPSS 25.0.

3. Results

3.1. EWC-DOM Reversed Glyphosate Stress on the Growth of Citrus Plants

As shown in Figure 1A, the growth of citrus seedlings under different treatments ranked from strong to weak as: E2 > E1 > G + E2 > G + E1 > Control > Gly. Quantitatively, compared to the control, the addition of EWC-DOM to the soil at 40 mg of DOC kg⁻¹ (E1) significantly increased the elongation and biomass of citrus seedlings by 50% and 77% for the shoots, and 45% and 69% for the roots, respectively (p < 0.05) (Figure 1B,C). Such positive regulation effects on citrus growth continued to strengthen with the increase of the EWC-DOM application rate to 80 mg of DOC kg⁻¹ (E2). Compared to the control, glyphosate treatment remarkably reduced the length (by about 20%) and dry weight (by over 40%) of citrus seedlings, which was drastically reversed through involving EWC-DOM to be even better than the control (p < 0.05). The elongation and dry mass of seedlings were approximately 1.17 and 1.15 times (G + E1) that of the control for shoots, and 1.21 and 1.23 times (G + E1) for roots, respectively. The growth of the above- and below-ground parts of seedlings in G + E2 was also enhanced by EWC-DOM. Their elongation and biomass were, respectively, 1.32 and 1.42 times the control for the shoots, and 1.32 and 1.41 times for the roots (Figure 1B,C).

3.2. EWC-DOM Adjusted Physiological Responses of Citrus Plants under Glyphosate Stress

Chlorophyll is such an important participant in photosynthesis, which plays a crucial function in the development and growth of plants. It was commonly adopted to monitor plants’ growth and nutrition status [31]. In our study, the chlorophyll contents of citrus seedlings from different treatments varied in the subsequent order: E2 > E1 > G + E2 > G + E1 > Control > Gly (Figure 2A), sharing the same trend as the growth of citrus plants (Figure 1). Concretely, the chlorophyll content of the citrus seedlings treated with 40 mg DOC kg⁻¹ and 80 mg DOC kg⁻¹ EWC-DOM was 1.27 and 1.40 times of the control, respectively, indicating that EWC-DOM addition could enhance plant photosynthesis via increasing their chlorophyll content (Figure 2A), thereby helping the seedlings thrive (Figure 1). Compared to the control, glyphosate exposure decreased the chlorophyll content of citrus plants by 23%. Whereas, astonishingly, the chlorophyll content of citrus plants suffering from glyphosate but in the presence of EWC-DOM increased rather than decreased, to 1.13 times (G + E1) and 1.20 times (G + E2) that of the control, respectively (Figure 2A).

The extent of membrane lipid peroxidation, reflected by TBARS content in plant tissues, is a trusted biomarker of the status of plants’ membrane damage [32]. The drop of TBARS in the shoots was 17% (E1) and 29% (E2) in comparison with the control, which was much steeper in the roots, up to 25.3% (E1) and 44% (E2), respectively (Figure 2B). However, in Gly treatment, the TBARS content of citrus plants increased significantly in comparison with the control, by 50% in the shoots and 77% in the roots (p < 0.05) (Figure 2B). As shown in Figure 2B, the involvement of 40 mg DOC kg⁻¹ and 80 mg DOC kg⁻¹ EWC-DOM specifically relieved the glyphosate-induced membrane lipid peroxidation in the roots by 17% and 27%, compared with 9% and 21% in the shoots.

3.3. Effect of EWC-DOM on the Activities of Detoxifying Enzymes in Citrus Seedlings under Glyphosate Stress

CPR is an indispensable component of the cytochrome P450s system, which transfers electrons from NADPH to P450s enzymes to maintain the performance of phase I metabolism pathways, the rate-limiting steps in the metabolism of foreign chemical substances in plants [33,34,35]. As shown in Figure 3A, the glyphosate-induced distinct rise (by 207% in the shoot and 90% in the root) in CPR activity detected in this study was drastically down-regulated via EWC-DOM involvement (by 23% (G + E1) and 35% (G + E2) in the shoots and by 13% (G + E1) and 22% (G + E2) in the roots (p < 0.05) (Figure 3A).

Laccases are another typical phase I enzyme, catalyzing single-electron oxidation of substrates not limited to phenolic substances in the presence of dioxygen [36] They are the favored choices for enzymatic bioremediation of organic contaminants, because of their wide range of substrates, together with their stable and powerful catalytic performance [37]. Figure 3B shows that laccase activity in the shoots and roots of citrus seedling was significantly increased by soil glyphosate, to 2.84 and 2.41 times that of the control, respectively (p < 0.05), indicating that laccase was effectively activated in response to glyphosate stress. Meanwhile, in the treatment of G + E1 and G + E2, laccase activity was markedly lower than that of the Gly group. Compared with the control, treatment with EWC-DOM at concentrations of 40 mg DOC kg⁻¹ and 80 mg DOC kg⁻¹ triggered a significant increase in laccase activities (Figure 3B), suggesting that EWC-DOM could invigorate the detoxification enzyme system of citrus seedlings to slow down the damage of citrus seedlings under stress conditions.

GST is another classic stress-tolerance enzyme working in phase II of plants, catalyzing the conjunction of the thiol group of tripeptide glutathione (GSH) to the electrophilic group of substances such as H₂O₂ and pesticides [38]. In the present study, compared with the control, GST activity rose by 117% in the shoot and 77% in the root under glyphosate stress. The change rate of GST activities was still fairly substantial, though more modest than CPR and laccase (Figure 3A–C). In seedlings treated with EWC-DOM alone, the activities of GST were significantly greater than the control, which leading to much stronger stress-defense capability, enabling them more leisure in the face of glyphosate stress. For this reason, GST activity in citrus seedlings treated with glyphosate in the presence of EWC-DOM was notably lower than that of the Gly treatment (p < 0.05) (Figure 3C).

As shown in Figure 3D, compared to the control, treatment with EWC-DOM at concentrations of 40 mg DOC kg⁻¹ and 80 mg DOC kg⁻¹ triggered a significant increase in UGT activities (by 14% and 31% in the shoots, and by 15% and 24% in the roots, respectively), resulting in expanded detoxification capacity to pesticides (p < 0.05). Among the six treatment groups, Gly treatment had the highest activity of UGTs in the shoots and roots of citrus seedlings, which was 2.07 and 1.75 times that of the control, respectively, to cope with glyphosate stress in plant tissues. Therefore, from the distinct reduction of UGT activity caused by the involvement of EWC-DOM, it can be inferred that EWC-DOM could alleviate the glyphosate-induced toxic effect on plants. As shown in Figure 3D, compared to the Gly group, the activity of UGTs in citrus seedlings treated with glyphosate in the presence of 40 mg DOC kg⁻¹ and 80 mg DOC kg⁻¹ of EWC-DOM decreased by 21% and 26.0% in the shoots, and by 10% and 20% in the roots, respectively (Figure 3D).

3.4. EWC-DOM Adjusted the Activities of Antioxidant Enzymes in Citrus Seedlings under Glyphosate Stress

SOD is considered to be plants’ first line of defense against oxidative stress, scavenging O₂−• through catalyzing its disproportionation into H₂O₂ and O₂ [39]. Next, H₂O₂, another major ROS, whether or not it originates from the disproportionation of O₂−•, would be cleaned up by special enzymes such as POD and CAT [40]. This study showed that the SOD activity in citrus seedlings from the E1 and E2 treatment groups was 1.25 and 1.37 times of the control in the shoots, and 1.13 and 1.22 times that of the control in the roots, respectively (Figure 3E). Similarly, EWC-DOM treatment alone also increased POD and CAT activities in citrus seedlings compared with the control. Thereinto, CAT activity increased by 25% (E1) and 49% (E2) in the shoots and by 19%–39% in the roots (Figure 3F,G). Class III POD appears in the cell wall and vacuole of plants, which exists in the form of a variety of isoenzymes with diverse functions, including the synthesis of lignin and consumption of H₂O₂ and Phenolics [41]. Herein, the increased magnitude of POD activities was more impressive than CAT, by 38% (E1) and 73% (E2) in the leaves and 22% (E1) and 54% (E2) in the roots, respectively (p < 0.05) (Figure 3F,G). From this result, together with the findings from the growth, chlorophyll content, and membrane lipid peroxidation status tests, it can be inferred that EWC-DOM was capable of helping plants to thrive so that they possess a superior antioxidase pool to resist aging induced by ROS.

In Gly treatment, to deal with glyphosate-induced oxidative damage, SOD activities were increased to 139% and 111% of the control in the shoots and roots of citrus seedlings, respectively, which were then down-regulated through the involvement of EWC-DOM by 29% (G + E1) and 36% (G + E2) in the shoots, and by 31% (G + E1) and 37% (G + E2) in the roots (Figure 3E). These results indicated that SOD could sensitively respond to the glyphosate-induced oxidative stress, and the involvement of EWC-DOM could alleviate glyphosate-induced ROS stress as reflected by the decreased membrane lipid peroxidation level (Figure 2B), thus, no longer requiring such high SOD activities.

Compared to SOD (Figure 3E), glyphosate exposure triggered much bigger gains in CAT activity, by 174% in the shoots and 143% in the roots (Figure 3F), to deal with the glyphosate-aggravated accumulation of H₂O₂, which partly generated from O₂−• disproportionation catalyzed by SOD. In this case, the effectively controlled ROS overload due to glyphosate by EWC-DOM is the logical reason for the significantly lower CAT activity in citrus seedlings treated with glyphosate in the presence of EWC-DOM than that in the absence of EWC-DOM. In Gly treatment, POD activities increased by 228% and 214% in the shoots and roots in comparison with the control (Figure 3G). Then, similar to SOD and CAT, the intervention of EWC-DOM resulted in a considerable drop in POD activity, indicating the alleviated glyphosate stress.

4. Discussion

In this paper, the application of EWC-DOM was fairly favorable for citrus seedling growth in soil with or without glyphosate, which could even help seedlings conquer the inhibitory effect of glyphosate on citrus seedling growth. This phenomenon might be contributed to the abundant nutrients in EWC-DOM eluted from EWC [30] which were conducive to soil microbial activity and plant growth, such as organic humus [42], enzymes [43], as well as nitrogen, phosphorus, and other microelements [44], thereby helping plants thrive on having a stronger immunity to external stresses.

Photosynthesis is the basis of plant growth [31]. Glyphosate is a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor that hinders the plant shikimic acid pathway, restraining the biosynthesis of related proteins and plant secondary metabolites, including photosynthesis-related compounds in photosystem II [45]. In our study, the participation of EWC-DOM can effectively restore decreased chlorophyll content due to glyphosate stress to be even slightly higher than that of the control. Phosphorus is an essential element for the growth and development of fruit trees. It is directly involved in various links of tree photosynthesis and the transformation and metabolism of chloroplast energy [46]. However, readily available P for plant uptake and utilization is usually limited in soil solution [47] but abundant in EWCs [48]. Thus, the strong reversal effect of EWC-DOM on a glyphosate-damaged chlorophyll pool in citrus seedlings should be attributed to its high content of available phosphorus from EWCs [46].

Membrane lipid peroxidation usually takes MDA as the final product, which will damage the structure and function of the cell membrane, thus affecting the normal operation of a series of physiological and biochemical reactions [32]. Our results suggested that EWC-DOM could help plants slow down the aging caused by lipid peroxidation of cell membranes [49]. As reported by Botten et al. [50], the roots of citrus plants were the more preferred site for glyphosate accumulation compared to the shoots; thus, the roots were more affected by glyphosate stress (77% > 50%). The suppressed root development (Figure 1) would reduce its ability to absorb and utilize nutrients from soil, thereby resulting in crop plants’ nutrient deficiency and stunting. From this perspective, EWC-DOM application was a sound symptomatic treatment to mitigate the negative effects of orchard weeding with glyphosate. The improved cell structure and cell membrane fluidity corresponding to alleviated membrane lipid peroxidation [51] would, in turn, benefit plants’ photosynthesis (Figure 2A) and growth (Figure 1).

Plant cells harbor the machinery of metabolic enzymes responsible for detoxifying extrinsic compounds, which were labeled as the “green liver” for their similar function to the liver in mammals [52]. Based on the diversity of their functions, these metabolic enzymes are classified into three categories, as the well-known phases I, II, and III, respectively [53]. Xenobiotics would undergo the transformation of chemical structure in phase I, followed by a combination in phase II into metabolites that are easily compartmented in phase III [54]. In this paper, backed by the promoted growth (Figure 1), increased chlorophyll content, and relieved membrane lipid peroxidation (Figure 2) of citrus seedlings using EWC-DOM, the elevated activities of CPR (Figure 3A) in citrus seedlings treated with EWC-DOM alone, in comparison with the control, were a good embodiment of the exuberant metabolism in plants, corresponding to a much stronger capability to detoxify foreign toxic substances. The sharp increase in CPR activity induced by glyphosate revealed that glyphosate should also be logged in the irritant list of CPR, which was previously documented as an inducible enzyme, sensitively responding to the stimulation of most exogenous substances [55,56]. EWC-DOM could ease glyphosate stress on plants through stimulating the vigorous metabolism of plants so as to minimize glyphosate damage to plant development. With or without external stress, EWC-DOM could elevate CPR enzyme activity, thus enhancing the detoxification capability of plants to foreign harmful substances and their adaptation ability to unfavorable environments. Indeed, the elevated enzyme activities in citrus seedlings treated with EWC-DOM alone or in combination with Gly demonstrated the exuberant metabolism in plants, thereby possessing strong capabilities to deal with abiotic stress.

Yet, for other typical phase I enzymes [36], there are few in vivo studies on the effect of laccase in plants under external pressure. Some studies have shown that laccase in rice is involved in the detoxification of atrazine and isoproterenol through a water incubation test [57]. Our study would help decode the role of laccase in living fruit trees in response to pesticide stress in the soil culture system. In our current study, EWC-DOM can reduce glyphosate-elevated laccase activity, possibly due to fewer glyphosate-related substrates demanding degradation by laccase. This might be ascribed to the reduced bioavailability of glyphosate by EWC-DOM, which could accelerate the degradation of glyphosate in soil or form a relatively stable complex with glyphosate, thereby making less glyphosate available for plant uptake [18].

GST can remove glyphosate and H₂O₂ accumulation induced in citrus seedlings and plays a crucial role in the detoxification of xenobiotic compounds in cells [19]. In our current study, EWC-DOM decreased the activity of stress-tolerant enzymes induced by glyphosate. Consistent with the findings of Jiang et al., our result suggests that there might be dissolved substances or microorganisms in EWCs which were conducive to reducing the bioavailability of soil glyphosate to plants [17]. In addition, as shown in Figure 3C, the GST activity of citrus seedlings in the roots was almost twice as high as that in the leaves, suggesting that the plant roots were directly exposed to pesticides and, thus, sustained much greater damage. Hence, the roots required higher enzyme activity to participate in pesticides detoxification.

UGTs, a superfamily of glycosyltransferases, are vital enzymes of phase II. They detoxify exogenous compounds through catalyzing their conjunction with UDP-nucleotides, among which, UDP-glucose is the most common sugar donor in plants [36]. UGTs could fulfill the structural modification and detoxification of pesticides through catalyzing the deprotonation of pesticide molecules [34,36]. Our results showed that, compared to the control, EWC-DOM treatment induced a significant increase in the activity of UGTs. Hence, we speculate that UGTs are involved in the process of metabolic glyphosate detoxification. While among the six treatment groups, the highest UGT activity was detected in the citrus seedlings of glyphosate treatment, the UGTs activity was higher in the belowground parts than in the aboveground parts (Figure 3D), indicating that plants activated UGTs in response to glyphosate stress, and EWC-DOM alleviated the toxic effects of glyphosate on plants.

The aggravated membrane lipid peroxidation detected in citrus seedlings under glyphosate exposure (Figure 2B) was commonly recognized as a consequence of reactive oxygen species (ROS) burst, a sensitive plant response to external stress that can lead to oxidative damage to plant tissues [58]. The antioxidant enzymes represented by SOD, POD, and CAT are responsible for the removal of the excessively accumulated ROS in plants to cope with the outbreak of ROS (including superoxide anion (O₂−•) and hydrogen peroxide (H₂O₂)) caused by exogenous toxic substances, such as pesticides [59,60].

SOD is a primary scavenger of O₂−•. Radicals, and both CAT and POD, are essential enzymes in combating the accumulation and toxicity of H₂O₂ in plants. The only difference is that CAT is almost exclusively dedicated to the dismutation of H₂O₂ into H₂O and O₂ [61]. The relatively higher activities of antioxidant enzymes in citrus plants treated with EWC-DOM alone than those of the control further supported our conclusion above, that EWC-DOM application could promote the sturdy growth of plants with a much more vigorous metabolism (photosynthesis and respiration, etc.), which in turn would produce more ROS to be detoxified via much higher activities of antioxidant enzymes. Or rather, EWC-DOM could promote the growth of citrus plants, which makes them have a higher basal metabolic rate. Of all the enzymes involved in this paper, POD was the most sensitive one to glyphosate stress, mainly for the cleanup of H₂O₂ accumulation exacerbated by glyphosate, with some for the synthesis of lignin to set up roadblocks against glyphosate [41]. In addition, the activities of all enzymes increased significantly with exposure to glyphosate alone, suggesting that a range of stress-tolerant enzymes were activated in plant cells in response to glyphosate stress; meanwhile, EWC intervention significantly reduced glyphosate-elevated enzyme activities, which was also the reason for the decrease of membrane lipid peroxidation and the increase of chlorophyll content.

5. Conclusions

EWC-DOM played the role of a nutrient-rich organic fertilizer to help the vigorous growth of plants, and thus possesses a more capacious detoxification and antioxidant enzyme pool, with a stronger ability to resist external stress such as glyphosate. As shown in our soil pot experiment, compared to treatment with glyphosate alone, the involvement of EWC-DOM could significantly raise the chlorophyll content in plant tissues, mitigate the degree of membrane lipid peroxidation of the seedlings, and promote plant growth (elongation and biomass) to be even better than the blank control. These results inspired us to further explore the causes from the perspective of plant stress resistance, which is characterized by a range of defense-related enzymes. We found that, as a water extract from EWCs, a well-known nutrient-rich organic amendment, EWC-DOM enriched water-soluble nutrients from EWCs that were readily absorbed and utilized by plants. It acted as a nutrient solution to help plants thrive and thus have a greater ability to cope with external coercion. When exposed to glyphosate stress of up to 12.15 mg kg⁻¹ together with EWC-DOM, the growth status of citrus seedlings was significantly better than that of the control group instead of being inhibited. Our data would be instructive for pesticide-contaminated soil remediation, the organic substitution of chemical fertilizers in agriculture, and the sustainable and healthy development of citrus production.