Zinc sulphate or zinc nanoparticle applications to leaves of green beans

For this study, plants of the green bean (P. vulgaris L.) cv. ‘Strike’ (short cycle variety, sensitive to salinity and adapts to greenhouse conditions) were grown in polyethylene bags filled with 3 kg of soil (sandy loam, pH 6.8, electrical conductivity 0.51 dS · m⁻¹, organic matter 1.5%, water saturation 48%) (Figure 1). The soil composition contained the micronutrients in mg · kg⁻¹: 0.22 Zn⁺², 0.4 Cu⁺², 5.4 Fe⁺² and 1.3 Mn⁺². Plants were raised in an experimental greenhouse in Chihuahua, Mexico (28°41′ 29.8″ N, 106°06′58.6″ W) with a mean temperature of 30 °C. Mineral nutrition was carried out using a solution prepared with distilled water and containing the macronutrients (mM): 7.5 NH₄NO₃, 1.0 K₂HPO₄, 1.05 K₂SO₄, 2.5 CaCl₂·2H₂O, 1.0 MgSO₄ and the micronutrients (μM): 44.6 MnSO₄·H₂O, 1.2 ZnSO₄·7H₂O, 0.185 CuSO₄·5H₂O, 0.06 (NH4)₆Mo₇O₂₄·4H₂O and 0.5 H₃BO₃ (Ponce-García et al., 2019).

Figure 1

We used a completely randomised experimental design with 10 replicates, where the experimental unit consisted of one plant. There were two Zn treatments and a control (without Zn). The first Zn treatment used an aqueous solution of ZnSO₄ (150 mg · L⁻¹, Merck^®, Germany) and the second an aqueous suspension of ZnO NPs (Investigación y Desarrollo de Nanomateriales S.A. de C.V., Mexico). Both treatments were applied before sunrise using a soft brush to the upper and lower surfaces of the first trifoliate leaves. To avoid any contact with the soil, each plant was sprayed separately, and a plastic film was used to cover the top of each pot before spraying. Foliar application was done every 10 days for a total of three applications. After physiological maturity of the plants, at 60 DAG, the samples were taken and separated into four parts: root, stem, leaf, and fruit and washed thrice with distilled water and 1% non-ionic detergent.

The material applied as Nfs was ZnO obtained by wet chemistry method in the form of wurtzite crystals with an average size of 50 nm with negligible contaminants, a purity level of 99.7% and a density of 5.61 g · cm⁻³ (Figure 2 and 3) (Ponce-García et al., 2019).

Figure 2

Figure 3

Fruit yield was determined at the end of the phenological cycle. The results are expressed in grams of fresh weight per plant (g · p⁻¹ FW). Biomass was determined according to the method indicated by Sida-Arreola et al. (2015). In each sampling, the fresh weight of leaflets, stems and roots were taken and dried in a Heratherm VCA 230^® (Thermo Scientific, USA) oven at 70 °C for 72 h. The results are expressed in grams of dry weight per plant (g · p⁻¹ DW).

Prior to the statistical analysis, Levene's test for homogeneity of variances was carried out for each group of data per variable (Sokal and Rohlf, 1995). Subsequently, the analysis of variance was carried out and when a significant treatment effect was detected, Tukey's multiple comparison of means test was applied (p ≤ 0.05). In all cases, the statistical program IBM SPSS Statistics 25 was used.

The application of ZnO NPs significantly increased the Zn²⁺ concentrations in the leaflets, roots, stems and pods with values of 94.66 mg · kg⁻¹, 80.30 mg · kg⁻¹, 87.18 mg · kg⁻¹ and 66.29 mg · kg⁻¹ DW, respectively (Figure 4). Except for the stems, the differences between the two treatments were significant. Ranked in order of efficacy, it can be expressed as ZnO NPs > ZnSO₄ > control. In this regard, Ponce-García et al. (2019), when evaluating the edaphic application of 100 mg · kg⁻¹ of Zn²⁺ (sulphate) in the green bean (P. vulgaris L.), reported a significant increase in the concentrations of Zn in the roots and leaflets with values of 31 mg · kg⁻¹ and 5 mg · kg⁻¹, respectively. In other crops, such as Coffea arabica L., applications of ZnO NPs increased the concentration of Zn²⁺ in the leaves but not in the stems or roots (Rossi et al. 2019). The plants used in this study did not show symptoms of Zn²⁺ deficiency, which could explain the foliar increase due to Zn micronutrient. On the other hand, it has been reported that the dissolution of ZnO NPs in deionised water is relatively slow, so in 24 h only 2% is dissolved, hence its effect can be better observed after 30 days (Singh et al 2017).

Figure 4

In this study, the application of ZnO NPs to the leaves indicates a significant concentration of Zn²⁺ to the pods, stems and roots. In contrast, in another study (Ponce-García et al. 2019), applications of 25 mg · kg⁻¹, 50 mg · kg⁻¹ or 100 mg · kg⁻¹ of ZnO NPs on the green bean ‘Strike’ showed enhancement in the roots but not in the stems. This variation in behaviour may be associated with the phenological stage, nutrient antagonism or applied dose, as indicated by Rossi et al. (2019). On the other hand, pods harvested from plants treated with ZnO NPs presented higher Zn²⁺ concentrations at 66.29 mg · kg⁻¹ (Figure 5). This suggests that ZnO Nfs could help in the biofortification of green bean of this variety. Similar results were reported for this variety by Ponce-García et al. (2019) with applications of 100 mg · kg⁻¹. In this sense, the use of ZnO NPs improves crop productivity by increasing the uptake efficiency, application accuracy and with less environmental impact (Shang et al., 2019). It has been shown that the level of uptake and translocation of ZnO NPs is associated with the applied dose and the phenological stage (Singh et al., 2017). However, little information is available on the physiological and biochemical effects of ZnO NPs application with this species.

Figure 5

Reduced synthesis photosynthetic pigment and accumulation are good indicators of stress or suboptimal growth conditions (Marzouk et al., 2019; Zargar Shooshtari et al., 2020; Ebrahimi et al., 2021). The concentration of photosynthetic pigments (chlorophyll, carotenoids and TAn) are presented in Table 1. The application of ZnO NPs increased the concentrations of chl a and chl b (15.40 μg · g⁻¹ and 11.64 μg · g⁻¹, respectively) with respect to ZnSO₄. Previous studies on the broad bean (Vicia faba L.) with foliar applications of 50 mg · L⁻¹ showed positive results with respect to the total chlorophyll concentration (Ghidan et al., 2020). It is known that most micronutrients such as Fe²⁺, Mn²⁺ Cu²⁺ and Zn²⁺ are involved in activation of various metabolic enzymes, including protein and chlorophyll biosynthesis (Sida-Arreola et al., 2015; Castillo-González et al., 2018; Souri et al., 2019; Aghaye-Noroozlo et al., 2019). These results contrast with those reported by Ponce-García et al. (2019) who, when applying ZnO NPs and chelates (25, 50 and 100 mg · kg⁻¹) in nutrient solution, report no significant effects. These authors point out an inverse behaviour between the doses of ZnO NPs and chlorophyll concentration, attributed to the stress generated by the possible toxicity caused by the application of these Nfs to the soil. In our study, no visible symptoms of Zn²⁺toxicity (wilting, curling, waving, chlorotic and necrotic spots) were detected (Pullagurala et al., 2018), which could be linked to the slow dissolution of ZnO NPs in deionised water (Rossi et al., 2019).

On the other hand, the concentrations of total carotenoids and anthocyanins were not affected by the application of ZnO NPs. Previous studies indicate a positive effect on plant productivity by increasing photosynthesis (Pullagurala et al., 2018; Marzouk et al., 2019); however, there are no specific references indicating changes in the concentrations of carotenoids and TAn for green beans. In this sense, it would be necessary to conduct evaluations with other doses, application times and growth stages to improve our understanding of the effects of NPs on the behaviour of accessory pigments.

In this study, the application of ZnSO₄ slightly increased the sucrose concentration (6.38 mg · g⁻¹), but it did not significantly exceed that observed in the treatment with ZnO NPs (5.53 mg · g⁻¹). The increase in photosynthetic activity is associated with the synthesis and activity of chlorophylls, which allows the accumulation of carbohydrates, including sucrose. Previous repor ts on green bean indicate that increase in the leaf concentrations of Mg²⁺, Fe²⁺ and Zn²⁺ affect carbohydrate accumulation (Sálcido-Martínez et al., 2020; Balafrej et al., 2020), which is associated with the participation of these micronutrients in the metabolism of proteins linked to the synthesis and accumulation of chlorophylls (Korkmaz et al., 2018). In general, a gradual increase in the concentration of this soluble sugar was observed in this research. This could be due to the simultaneous need to accumulate and translocate sucrose as a source of carbon and energy to the shoots and pods, as part of the overall growth and development (García-Caparrós et al., 2021).

On the other hand, treating leaflets with ZnSO₄ or ZnO NPs did not affect the concentrations of TP or TFl. To date, there are no reports comparing the effects of ZnO NPs with ZnSO₄ on the accumulations of bioactive compounds in green beans. However, according to our results we infer that the application of ZnO NPs and ZnSO₄ did not significantly modify the physiological and biochemical mechanisms of the plants. The small differences found can be associated with the solubility characteristics of ZnSO₄, which when applied to leaves can drop rapidly; therefore, the bioavailability of Zn²⁺ ions over a prolonged period is uncertain (Doolette et al., 2018). In addition to its physical properties (size, structure and particle shape), previous studies with foliar applications of ZnO NPs varying between 1,000 mg · L⁻¹ and 2,500 mg · L⁻¹ on Brassica nigra and Capsicum chinense indicate a significant effect on FT and TFl concentrations (Zafar et al. 2016; García-López et al., 2019). On the other hand, Abbasifar et al., (2020) when applying 1,000 mg · L⁻¹, 2,000 mg · L⁻¹ or 4,000 mg · L⁻¹ report a significant increase in the concentration of flavonoids in basil leaves (Ocimum basilicum L.). However, it is important to note that the ZnSO₄ and ZnO NPs doses applied in these studies exceed those used in our study by 10 times. According to Zafar et al. (2016), these could induce oxidative stress and stimulate the synthesis of non-enzymatic compounds (phenolic compounds, flavonoids, carotenoids, among others). It is known that under optimal conditions, Zn²⁺ plays an important role in the synthesis and activation of superoxide dismutase (SOD), the main enzyme associated with the removal of reactive oxygen species (ROS) (Korkmaz et al. 2018). This behaviour changes radically when the concentration of this micronutrient is at phytotoxic levels, causing oxidative stress (García-López et al., 2019). In this sense, the response to foliar Zn²⁺ application is a function of the species, nutrient status, phenological stage, dose supplied and number of applications (Shang et al., 2019; Abbasifar et al., 2020).

The applications of ZnSO₄ and ZnO NPs showed no effects on AC performance. This result was similar to that observed with foliar concentration of TFl (Table 2). In contrast, significant increases in AC values have been reported in other species, including basil (O. basilicum L.) (Abbasifar et al., 2020), potato (Solanum tuberosum L.) (Korkmaz et al., 2018) and tobacco (Nicotiana tabacum L.) (Mazaheri-Tirani et al., 2019). Korkmaz et al. (2018) report significant variations in AC resulting from the interaction between dose and time of application, suggesting that Zn²⁺ plays a key role in the synthesis and activation of the plant antioxidant system in mitigating oxidative stress (Balafrej et al., 2020).

The applications of ZnSO₄ and ZnO NPs showed no significant variation in relation to sucrose concentration in the pod and seeds of green bean. However, TFl concentrations were significant for the seed (Table 2). These results may be related to the dose and number of Zn²⁺ applications. Usually, the application of Zn²⁺ increases the concentrations of nutritional and bioactive compounds, however, information on the effects of applications of ZnSO₄ and ZnO NPs on the concentrations of sucrose and TFl in pods and seeds of green bean is limited. One of the factors offered to explain the high consumption of green beans is their high nutritional value, mineral, fibre, protein, carbohydrate and vitamin contents (Minnocci et al., 2018). However, this crop is highly susceptible to Zn²⁺ deficiency in soils with high carbonate contents (Doolette et al., 2018; Souri and Aslani, 2018). There is evidence only for leaves, as indicated by Fatollahpour-Grangah et al. (2020); when applying ZnO NPs (1.5 mg · L⁻¹) on pinto beans – cvs ‘Coosha’, ‘Ghaffar’ and ‘Sadri’ – significantly increased the concentrations of soluble sugars. Abbasifar et al. (2020) report a significant increase from 11.09 mg · g⁻¹ to 25.27 mg · g⁻¹ DW in leaf concentration of TFl by applying 1,000 mg · L⁻¹ and 4,000 mg · L⁻¹ of ZnO NPs, respectively.

Plant defence systems include antioxidant enzymes (peroxidases, SOD, catalase and ascorbate peroxidase) and antioxidant compounds (phenols, flavonoids, glutathione, carotenoids, among others) that can inhibit or reduce the production and effects of ROS (Ahmadi et al., 2019; Abbasifar et al., 2020; Hatamian et al., 2020). In this study, ZnO NPs sprays affected TP values in pods and seeds; however, only in the pods was the AC significantly affected with both Zn²⁺ sources (Table 2). Recent studies have demonstrated the positive effects of Zn²⁺ application in the form of ZnO NPs and conventional ZnO on the AC of tomato (Solanum licopersicum L.) (Akanbi-Gada et al., 2019) and habanero pepper (Capsicum chinense Jacq.) fruits (García-López et al., 2019). On the other hand, it has been observed that the application of ZnO NPs alone promotes the generation of ROS near the particle surface; however, their lifetime is short and they are eliminated by antioxidant agents (Pedruzzi et al., 2020). In this experiment, a low dose of Zn²⁺ (ZnSO₄ and ZnO NPs) was used, which did not allow observation of the characteristic symptoms of phytotoxicity (necrosis and reduction of photosynthetic activity in leaflets), which confirms the importance of dose, number of applications and plant species to obtain positive effects on the physiology and biochemistry.

P. vulgaris L. is one of the most important crops for human nutrition worldwide (Ewais et al., 2017). In this study, applications of ZnSO₄ and ZnO NPs significantly increased both biomass (79.10 g · p⁻¹ and 84.70 g · p⁻¹ DW) and yield (55.64 g · p⁻¹ and 53.80 g · p⁻¹ FW) compared with the control (Figure 3). In this regard, it is important to note that Zn²⁺ is a micronutrient that plays important roles in photosynthesis, because it alters the chlorophyll concentrations and modifies (Ewais et al., 2017; Pullagurala et al., 2018; Shang et al., 2019). These results agree with those of Ponce-García et al. (2019) who, when evaluating soil applications of 25 mg · L⁻¹ (ZnO NPs) and 50 mg · kg⁻¹ (ZnSO₄) reported increases of 42% and 35% of biomass respectively, compared with the control. On the other hand, the yield values were found to exceed those reported by Ponce-García et al. (2019) when applying soil doses of 50 mg · kg⁻¹ and 25 mg · kg⁻¹ of ZnSO₄ and ZnO NPs, respectively. It has been reported that the efficiency of NPs is associated with the particle size, chemical structure, surface area covered and application rate (Aguilar-Carpio et al., 2018; Rossi et al., 2019; Ghidan et al., 2020). However, in this study the application of ZnO NPs and ZnSO₄ showed similar behaviours. These behaviours may be associated with the solubilities and stabilisation of the acidic pH of the solution presented by ZnSO₄ (Minnocci et al., 2018).