Effects of Pesticides on Physiological, Anatomical, Biochemical and Molecular Characteristics of Tomato (Lycopersicon esculentum)

Abstract

Application of synthetic pesticides is a common practice in agriculture around the globe and this trend is increasing with the passage of time. Pesticides are highly toxic substances that besides providing protection against pests’ cause toxicity in host crop. Their toxicity may not be absolutely confined to the target pests but can adversely affect different processes in the non-target host plants. Pesticide-induced stress can affect non-target plants by reducing germination potential, hindering in the biomasses, affecting the production of photosynthetic pigments, through elevated levels of reactive oxygen species (ROS) responsible for detrimental effects on cell metabolism, biochemical and other physiological activities. In response to oxidative stress, plant activates antioxidant defence system consisting of both enzymatic and non-enzymatic components. Furthermore, molecular changes may occur in response of pesticides stress in tomato. Tomato (Solanum lycopersicum L.) is an important crop but is often compromised with lower yield due to pest attacks. Over application of pesticides indiscriminately or due to unskillful uses, affects the growth of vegetables crops. In the present study, the effect of over application of three commonly used pesticides (emamectin benzoate, alpha-cypermethrin and imidacloprid) on seed germination, seedling vigor, photosynthetic pigments and oxidative stress were evaluated. In addition, cell viability, cell injury, total soluble sugar (TSS) and total soluble proteins (TSP) were also estimated. Antioxidant activities were measured i.e. superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) peroxidase (POD), ascorbate peroxidase (APX) and proline. For assessing the oxidative stress, hydrogen peroxide (H2O2) levels were analysed as ROS, lipid peroxidation was measured in term of thiobarbituric acid reactive substances (TBARS) as membrane damage. For anatomical studies, the diameter of tomato shoots and roots, stomatal indices, length and width of stem and root cortical cells and xylem and phloem cells were determined using scanning electron microscopy (SEM). The DNA damage and changes in the protein profiles were assessed through RAPD and SDS-PAGE, respectively. For the RAPD analyses, 14 RAPD primers were used to generate polymorphic band profiles of untreated and treated tomato seedlings. The obtained results revealed that seed germination was decreased by the pesticides and this effect was more prominent at early stages of exposure. All the tested pesticides reduced the growth of tomato when applied in higher concentration than the recommended dose, but at lower doses the pesticides had some stimulatory effects on growth as compared to the control. A similar effect of pesticides was observed on the photosynthetic pigments, i.e. a decrease in pigments concentrations was caused by higher doses but an increase was observed at lower doses of pesticides. The calculated EC50 values for different parameters revealed the lowest EC50 values for emamectin (ranged as 51–181 mg/L) followed by alpha-cypermethrin (191.74 – 374.39) and imidacloprid (430.29 – 1979.66 mg/L). A comparison of the obtained EC50 values for different parameters of tomato with the recommended doses revealed that over application of these pesticides can be harmful to tomato crop. Pesticides application at higher concentrations significantly elevated ROS levels and caused membrane damage by the formation of TBARS, increased cell injury and reduced cell viability both in root and shoot tissues compared with non-treated plants. Moreover, a gradual decrease in the levels of TSS and TSP was observed in plants subjected to increasing doses of pesticides. To cope with pesticide-induced oxidative stress, a significant increase in levels of antioxidants was observed in the plants exposed to higher doses of pesticides. Shoot tissues responded more drastically by producing higher levels of antioxidants as compared to root tissues indicating the direct exposure of shoots to foliar application of pesticides. The obtained results indicated that application of pesticides reduced the stomatal index, especially in samples exposed to higher levels of pesticides as compared to the untreated control. A significant decrease in the length and width of stomatal guard’s cells and epidermal cells was observed in the pesticides-treated seedlings. Roots showed a higher sensitivity than stem to pesticide stress. Statistical analysis revealed that in pesticides treated tomato seedlings, a significant reduction occurred in shoot and root diameters. Moreover, the length and width of epidermal and cortical cells as well as dimeters of xylem and phloem’s cells were affected significantly in the roots only. Taken together, results of this study suggested that exposure of tomato seedlings to pesticides above the recommended doses affected the morpho-anatomical features as evident by reduction in stomatal number, low stomatal index, reduced length and width of epidermal cells in leaves, stems and roots. Furthermore, pesticides exhibited distinctive polymorphisms based on size, appearance and disappearance of DNA and polypeptide bands as compared with the control. Polymorphisms and genomic DNA template stability (GTS) which is a qualitative measure of DNA banding patterns compared with control RAPD profiles, revealed that higher doses of pesticides have genotoxic potential. Among the pesticides concentrations, 4X and 2X doses of imidacloprid and 4X dose of both emamectin and cypermethrin resulted in the disappearance of polypeptide bands. In addition, a dendrogram constructed using cluster analysis based on genetic similarity coefficients, grouped the treatments in different branches indicating that samples exposed to higher doses clustered separately. In a few cases these pesticides were found toxic even at the recommended doses. Pesticides application at higher concentrations significantly altered proteins and DNA banding patterns in tomato. Taken together, these results strongly suggested that the application of pesticides above the recommended dose can provoke the state of oxidative stress that can cause oxidative damages and in turn altered the DNA and protein profiles based on RAPD and SDS-PAGE analysis, respectively.