Current studies exploring the adverse phytotoxic effects of exposure to pesticides have boomed. Triazole fungicides which may pose exposure risk to non-target crops, however, has so far received very insufficient attention. In today’s agroecosystem, fungicides and salt can be found simultaneously in soil, and can cause complex contamination to the soil environment, Consequently, combined pollution with fungicides and salt is a realistic scenario for crops. They, likely cause ecological risks (Touzout 2023). Therefore, evaluating the phytotoxic effects induced by DIF and/or NaCl exposure in plants and the underlying mechanism may have useful implications.
As shown in Table 1, the results described in this investigation confirm the phytotoxicity of the fungicide already reported for wheat plants (Liu et al. 2021a). This is evidenced by the impairment of seedling growth observed after 7 days of DIF exposure with a dose corresponding to field recommended concentration. The exposure to DIF resulted in a significant decrease of shoot fresh weight and length, whereas the root growth showed a similar trend of shoot growth (Table 1). A similar reduction in growth after exposure of plants to pesticides was reported in several studies such as the work of Bakshi et al, Barchanska and co-authors, and the work of Dong and colleagues (Bakshi et al. 2023; Barchanska et al. 2023; Dong et al. 2023; Jan et al. 2023; Kudrna et al. 2023; Li et al. 2023). However, further researches should be carried out to precisely establish the sensitivity of non-target crops to pesticides. NaCl stress induces detrimental effects in shoots and roots fresh weight and length, which can affect the physiological activities of seedlings and inhibit tomato growth (Dzinyela et al. 2023). The biometric attributes are an important parameter to estimate the effect of stressor factors on growth and physiological performance in plants. The decrease in plants biomass and length induced by salt stress was previously documented (Wang et al. 2023). This could be associated with impaired ion balance, oxidative injury induced by excessive ROS, a decline in nutrient ions and osmotic stress (Iram et al. 2023). In the current study, our growth parameters results indicated that co-contamination of DIF and NaCl significantly stunted the root growth and decreased the biomass of shoots in tomato seedlings. Thus, the result suggested that combined exposure to DIF and NaCl impaired tomato seedlings. Similarly, pesticide and salt stress resulted in a significant reduction in the growth of rice plants (Sharma et al. 2015). In addition, co-exposure to glyphosate and copper had a synergistic influence on the growth of Salvinia natans (Liu et al. 2019). Our study demonstrates for the first time the biological responses of the non-target crop species S. lycopersicum after co-exposure to DIF fungicide and NaCl stress. However, this scenario needs to be explored in future studies using omics approaches like metabolomics.
Photosynthetic pigments such as chlorophyll and carotenoids are vital for photosynthesis, and their levels are used as a biomarker and indicator of photosynthesis status of a plant under xenobiotic exposure (Soares et al. 2020). In this study, separate and combined exposure to DIF and NaCl had a significant adverse effect on chlorophyll a and b content in tomato leaves compared to the control, which is consistent with earlier study (Liu et al. 2021a; Askari et al. 2023). DIF exposure and NaCl stress reduced the chlorophyll content to a great extent. This may be due to the degradation and/or reduction in the formation of photosynthetic pigments caused by the over-accumulation of ROS induced by pollutants (Touzout et al. 2021a; Iram et al. 2023). A decrease in chlorophyll contents may result in an inhibitory effect on tomato seedlings growth (Tables 1 and 2). Interestingly, combined treatment inhibits the chlorophyll a content less severely than the DIF and NaCl alone treatments, suggesting their antagonistic effects on chlorophyll a content. In contrast, the degree of chlorophyll b inhibition under combined stress was greater than those of DIF and NaCl alone stressed, suggesting the synergistic effect of DIF and NaCl co-exposure on chlorophyll b content (Table 2). The synergistic or antagonistic effects on pigment in the leaves imply a potential interaction between DIF and NaCl exposure. Likewise, Li et al. (2018) who reported antagonistic effect of diclofop-methyl and silver nanoparticles co-contamination on chlorophyll contents in Arabidopsis thaliana. In agreement with our results, the synergistic effect of co-exposure to higher concentrations of glyphosate and copper on the chlorophyll contents of Salvinia natans was also stated by Liu et al. (2019). These different results of the combined phytotoxicity of contaminants may be related to type, concentration, application, and diversity in experimental and plant materials (Touzout et al. 2021b). Further investigations are needed to explore the interactive (synergistic/antagonistic) effects of pesticides and salinity on the photosynthetic pigments. Accessory pigments like carotenoids play a key role in plant tolerance to abiotic stress such as salt (Askari et al. 2023), heavy metals (Gill and Tuteja 2010), and pesticides (Sharma et al. 2019b). In the present study, the increase in carotenoids may be helpful for tomato seedlings to adapt to the stress induced by DIF and/or NaCl exposures (Table 2). In agreement with the present observation, a significant increase in carotenoids contents was observed under salt in Silybum marianum (Zahra et al. 2022) and chlorpyrifos organophosphorous insecticide in Brassica juncea (Bakshi et al. 2023).
Oxidative stress is a frequently observed phenomenon in plants exposed to pesticides (Sharma et al. 2019b) and slat toxicity (Dzinyela et al. 2023). However, most studies focused on the oxidative injury under the single pollutant exposure, but few studies examined the oxidative stress in non-target crops under combined exposure. MDA is a by-product of lipid peroxidation which can be an indicator of oxidative stress degree in cells (Gill and Tuteja 2010). In this study, the MDA level increasing significantly following DIF and NaCl exposures. Increased MDA content, provides that the generation of lipid peroxidation is stimulated. Several investigations in the literature found that plants often raise their MDA content to withstand different environmental stresses (Soares et al. 2019b, 2021; Talaat and Todorova 2022). However, combined exposure showed antagonistic effects on lipid peroxidation. This was because that DIF and NaCl could interact in cells and stimulate the activity of antioxidant enzymes, thereby decreasing oxidative damage and enhancing the detoxification of ROS (Mittler 2017). The originality of this work lies with the assessment of the biochemical targets on tomato seedlings exposed to fungicide and salt toxicity simultaneously. High contents of H2O2 were observed after DIF and NaCl exposures (Fig. 1B). Previous studies have reported that exposure to DIF and NaCl alone can induce the generation of H2O2 in plants (Liu et al. 2021a; da Silva Viana et al. 2023). Also, chlorothalonil treatment significantly enhanced H2O2 concentrations in tomato leaves (Peng et al. 2023). Furthermore, we also confirm an increase of lipid peroxidation after DIF and NaCl exposures (Fig. 1A), indicating oxidative injury, which represents a high threat to maintain cellular hemostasis that protects against the influences of xenobiotic agents (Soares et al. 2019a). Similarly, an elevated content of MDA was highlighted after exposure to insecticide voliam (VOL) and/or NaCl by tomato, and that xenobiotic exposure can lead to oxidative stress (Touzout 2023). Thus, it can be suggested that biochemical analysis would be useful to understand the toxic mechanisms underlying the interaction between DIF fungicide and NaCl toxicity in tomatoes.
Plant possesses a sophistical antioxidant defense system, in which CAT and APX are key H2O2 scavenging antioxidant enzymes. These two enzymes work together to keep H2O2 at a low and signaling level, thereby improving plant stress resistance (Gill and Tuteja 2010; Parvin et al. 2019). In this study, we found that the CAT and APX activities of tomato leaves were significantly inhibited compared to the control group, as the fungicide DIF and NaCl induced oxidative stress that exceeded the tolerance of seedlings, thus hindering antioxidant enzyme catalytic activity (Mittler 2017). The consistent inhibition of CAT and APX in the seedlings exposed to DIF or NaCl single stress resulted in a high accumulation of H2O2 and ROS which could not be efficiently scavenged and detoxified. Pesticide exposure mediated inhibition of APX and CAT has been reported previously (Jan et al. 2023; Peng et al. 2023). Importantly, the co-contamination of DIF and NaCl effectively alleviates the inhibition of CAT and APX activities induced by fungicide and salt stress, restoring to some extent the antioxidant defense system (Choudhury et al. 2017). Elevated maintenance of the APX activity following combined exposure clearly validated our earlier observation regarding H2O2 accumulation in the seedlings subjected to DIF and NaCl co-contamination (Fig. 1B and 2A).
Sulphur-containing molecules (Thiols) contribute to plant biotic/abiotic stress tolerance (Zhang et al. 2017). GSH and GST are vital for the elimination of excessive ROS and the biotransformation of xenobiotics (Noctor et al. 2012; Zhang and Yang 2021). GSH is an intracellular antioxidant non-protein thiol that functions as a substrate for glutathione-s-transferase in the detoxification of pesticides (Hernández et al. 2015). Leaves thiols profiles in tomato seedlings fluctuated following single and combined exposure to pollutants (Fig. 3). The GSH content decreased by individual DIF exposure, but increased by NaCl alone and in combination with DIF exposures (Fig. 3B). The decrease in GSH content by DIF exposure can be explained by the fact that more GSH binds to hydrophobic substrates to detoxify DIF fungicide (Yu et al. 2022). The rise in GSH amount can be a biochemical prevention strategy for oxidative damage to leaf cells (Noctor et al. 2012). The present results corroborate previous findings on bitter gourd under salinity (Iram et al. 2023), who reported a marked enhancement in GSH content in leaf tissues. GST contains a series of enzymes that play an important role in removing ROS and the metabolism of organic chemicals (Zhang and Yang 2021). The GST activity under DIF single treatments significantly increased, which was also observed under NaCl alone exposure (Fig. 2D). Consistent with the results of previous research, the glutathione detoxification pathway “green-liver” actively participated in the chlorothalonil detoxification process in tomato seedlings (Shan et al. 2022). Interestingly, our results indicated that DIF and NaCl co-contamination may increase GST activity more than both pollutants single exposure, so that more GST is involved in the detoxification of combined contamination (Touzout 2023; Wu et al. 2023). Besides GST, Peroxidase (POD) plays an important role in xenobiotic detoxification and is regarded as a biomarker of phytotoxicity (Liu and Wu 2018). We found that the activity of POD in tomatoes was significantly induced after pollutant exposure (Fig. 2D), suggesting that exposure to DIF and/or NaCl may disturb redox status in the leaves, and may even cause phytotoxicity. Touzout et al. (2021b) reported similar results in Solanum lycopersicum under IMI and/or Cd. Further research using molecular tools regarding the co-contamination effects of pesticides and salt on antioxidant and detoxification enzymes are essential to explore the potential underlying mechanisms.
Proline is an essential osmoregulator principally involved in maintaining the cell's turgor, stabilizing membranes and osmotic adjustment (Soares et al. 2019a). In this study, when applied individually, DIF and NaCl exposure enhanced proline accumulation, suggesting that the cells were incapable to maintain the osmotic homeostasis (Fig. 3). This increase in proline level enhanced osmotic stress tolerance in tomato seedlings and was also reported in rice under salt stress (da Silva Viana et al. 2023) and cucumber plants under difenoconazole exposure (Liu et al. 2021b). Iram et al. (2023) also reported that proline plays a crucial role in osmotic adjustment, maintaining the cell's turgor and stabilizing membranes, increases saline and alkaline stress tolerance in bitter gourd (Momordica charantia). However, upon exposure to combined stress, the proline level reduced to a level which was even lower than that in the DIF or NaCl exposed seedlings, but higher than that in the control seedlings (Fig. 3). This was probably due to the co-inhibitory effects of DIF and NaCl on proline biosynthesis enzymes and gens (Gill and Tuteja 2010).
To further study the phytotoxic effects of individual and combined exposure to DIF and NaCl on the tomato seedlings, the secondary metabolism was investigated. Secondary metabolites have an indispensable role in the regulation of the growth and the adaptation of plants to their environment, and the changes in their accumulation may alter the physiological status of the plants (Hernández et al. 2009; Sharma et al. 2019a). In the current study, the total phenolic and flavonoid contents significantly increased in the single-exposure group, and a similar trend was observed in co-exposure group (Fig. 4). An increase in secondary metabolites levels may stimulate the detoxification process of H2O2 and thus could protect cell from lipid peroxidation (Wu et al. 2023). Similar to our studies, Mohamed and Akladious (2017) reported that the flavonoid content increased in cotton plants treated with Flosan, Hemixet, and Maxim fungicides. In addition, the total phenolic contents of the white bean plants increased after exposure to salinity stress (Askari et al. 2023). At the enzymatic level, the activity of PAL increased in the leaves of the tomato seedlings after exposure, with a stronger degree of increase for co-exposure than for single exposure. PAL is the most dominant key enzyme of plant phenolic biosynthesis (Kováčik and Bačkor 2007), and their enhancement stimulates the phenylpropanoid pathway to produce phenols and flavonoids (Ahammed et al. 2013), thereby causing ecological adaptation or tolerance of the tomato seedlings to xenobiotic stress. Enhanced PAL activity is also reported in tomato leaves under PHE and/or Cd exposure (Ahammed et al. 2013). Interestingly, after tomato seedlings were exposed to DIF + NaCl, the activity of PAL and the accumulation of flavonoids increased much higher than those of single DIF and NaCl exposure, suggesting that phenylpropanoid pathway was more stimulated under combined pollution (Dong et al. 2023). Likewise, Touzout et al. (2021b) reported that imidacloprid (IMI) and/or cadmium (Cd) stimulated the PAL activity and increased the flavonoid contents in Solanum lycopersicum. In conclusion, this study suggests that exposure to DIF and NaCl in tomato stimulate the phenylpropanoid pathway, leading to increase secondary metabolites in the seedling leaves and that co-exposure stimulate the phenylpropanoid pathway on the leaves more than lonely exposure.