Effect of foliar application of zinc on annual productivity, foliar nutrients, bioactive compounds and oxidative metabolism in pecan

The research was carried out during the 2019 and 2020 production cycles on young (7-year-old) pecan [Carya illinoinensis (Wangenh.) K. Koch] cv. ‘Western Schley’ trees, established on native rootstocks, planted at 10 × 10 m spacing (100 trees · ha⁻¹). The orchard is located in Delicias, Chihuahua, Mexico (28°20’46.4”N, 105°34’03.3”W) at an altitude of 1,162 m; here, the mean annual temperature is 27 °C and the mean annual rainfall is 300 mm. The orchard is on a calcareous soil (Xerollic Calciorthid), has an arable layer of 0–35 cm with a pH of 7.8 in a 1:1 v:v soil:water mix, 0.95% organic matter, 20.0% total CaCO₃, 1.3 dS · m⁻¹ electrical conductivity (EC), 8.8 mg · kg⁻¹ NO₃-N and 0.54 mg · kg⁻¹ DTPA-extractable Zn. The trees used in this study had not previously received any Zn treatment. Mineral nutrient supply was by surface application of dry fertiliser (120 N: 100 P₂O₅: 96 K₂O). Standard commercial practices for weed control and irrigation scheduling were followed throughout the experiment.

Four different treatments were applied in a completely randomised design with 10 replications per treatment (40 trees in total). Each tree was one experimental unit. Tree height was 9.0 ± 0.5 m, and trunk girth was 55 ± 10 cm. All the spray solutions used contained 200 mg o L⁻¹ Zn (3.06 mM) and included the proprietary product NZN [nitrazinc, containing Zn(NO₃)₂ and UAN fertiliser (Smith and Storey, 1979); Tessenderlo K, Inc., Phoenix, AZ, USA]; ZnSO₄ (ZnSO₄·7H₂O reagent grade); Zn-EDTA (sodium salts and reagent grade ZnSO₄·7H₂O were added to make Zn(II)-EDTA); and ZnO NPs. The ZnO was obtained using wet chemistry as wurtzite crystals, of average size 50 nm with no contaminants (purity: 99.7%) and density 5.61 g · cm^-3 (Figures 1 and 2). In all formulations, 0.1% urea was added as the carrier ion and 100 mg · L⁻¹ of Tween® 20 (Thermo Fisher Scientific™, Waltham, MA, USA) was used as the non-ionic surfactant. The pH of the solutions was adjusted to 6.5 with HCl to facilitate foliar uptake in formulations with metallic nutrients (Ojeda-Barrios et al., 2014). The Zn solution was applied between 0600 hr and 0900 hr, using a 25-L motorised backpack fertiliser applicator, six times per season (2019 and 2020) – on 7 April; 6, 16 and 30 May; and 11 and 26 June. In each application, the solution was sprayed to run-off using 17 L per tree (2019) and 20 L per tree (2020).

Figure 1.

Figure 2.

Approximately 50 pairs of leaflets were collected from each tree, from the middle of the canopy, on 20 July (2019 and 2020). This timing was during the growing season and was approximately 215 days after budding, in the water stage of the nut. Leaflets were collected by pooling samples from the four cardinal directions (north, south, east and west) and from both vegetative and fruiting shoots. Collected materials were without obvious mechanical damage, pests or diseases. Nut collection was carried out in the last week of November by mechanical vibration of the trees.

Extraction and quantification of glucose, fructose, sucrose and starch were carried out according to the method described by Ojeda-Barrios et al. (2022).

Briefly, a sample of 1 g of fresh tissue was taken and homogenised twice, first with 5 mL of 95% aqueous ethanol (v:v) and next with 70% aqueous ethanol (v:v). The mixture was centrifuged at 5,500 g for 10 min at 4 °C. Next, 0.1 mL of the supernatant was taken, and 3 mL of anthrone solution was added. The mixture was placed in a water bath at 4 °C for 10 min and, after cooling, the absorbance was measured at 650 nm. For the determination of starch, the dry residue of the extraction was taken and incubated in acetate buffer (4.5 M), 0.5% α-glucoamylase (w:v) and water at 37 °C for 48 hr. The results are expressed in milligrams per gram of fresh weight (FW).

The performance components were determined according to Mexican Standard NMX-FF-084-SCFI-2009. The weight of the harvested nuts (fruits) was obtained using a Combo-Rhino-122 balance (Rhino®, Mexico City, Mexico) with a sensitivity of 0.1 g. Yield data are expressed in kilograms per tree. For the number of nuts per kilogram, nuts (1 kg) were randomly selected and counted. Next, 300 g of nuts were selected as a sub-sample, and the shells were removed and discarded. The kernels (the edible part) were weighed. Kernel percentage was obtained as the ratio of kernel weight divided by sub-sample weight × 100.

The leaflets were transported for analysis to the Plant Physiology Laboratory at Universidad Autonoma of Chihuahua, Mexico. Extraction and quantification of nutrients were carried out using the method published by Cruz-Álvarez et al. (2020). Briefly, a triple wash was carried out in tap water, then in 4 N HCl and finally in deionised water. Surface moisture was completely removed from the leaflets at room temperature, and the leaflets were then dried in a Heratherm VCA 230® oven (Thermo Scientific, Waltham, USA) at 75 °C for 24 hr. Each sample was homogenised in a Willey R-TE-650/1 mill with a 1 mm mesh (Tecnal, São Paulo, Brazil). The extraction and quantification of total-N and total-P were carried out by the Kjeldhal method (Novatech®, USA and Micro Kjeldahl Labconco ®, USA) and by the ammonium metavanadate method (NH₄VO₃) (Thermo Scientific™), respectively. The extraction of K⁺, Ca²⁺, Mg²⁺, Fe²⁺, Mn²⁺, Cu²⁺ and Zn²⁺ was by digestion in 25 mL of a triacid mixture (HNO₃, HClO₄ and H₂SO₄; 10:10:25) on a hot plate, under a fume hood. Analyte quantification was carried out using an Analyst 100^® atomic absorption spectrophotometer (PerkinElmer®, Waltham, MA, USA). The results are reported as grams per kilogram DW (for macronutrients) and as milligrams per kilogram DW (for micronutrients).

The extraction and quantification of the photosynthetic pigments were carried out according to the method described by Wellburn (1994). Briefly, the samples were placed in flasks with 100 mL of 80% (v:v) acetone. The absorbance values at 665, 653 and 470 nm were recorded using a Lambda 25® ultraviolet (UV)–visible spectrophotometer (PerkinElmer). The results are expressed in milligrams per kilogram FW.

The total phenol (TP) content was determined using the Folin-Ciocalteu method (Waterman and Mole, 1994) with slight modifications. Briefly, 0.5 g of sample was taken and homogenised with 5 mL of 70% (v:v) absolute ethyl alcohol. Next, an aliquot (50 μL) of this solution was taken and mixed with 7.95 mL of distilled water and 500 μL of the Folin–Ciocalteu (2N) reagent, then shaken for 1 min with a digital vortex (Thermo Scientific^®) and left to rest for 8 min. Then, 1.5 mL of 20% (w:v) Na₂CO₃ was added, stirred and left to settle for 2 hr in total darkness at room temperature (22 ± 1 °C). Absorbance values were recorded at 760 nm with a Lambda 25® UV-visible spectrophotometer (PerkinElmer). A standard curve of gallic acid (Sigma Aldrich, St. Louis, MO, USA) was constructed with concentrations of 0, 20, 40, 60, 80 and 100 mg · L⁻¹. The results are expressed in milligrams of gallic acid equivalents (GAE) per gram FW. The total flavonoid (TFl) content was determined using the method of Loizzo et al. (2016) with slight modifications. Briefly, 5 mL of methanol was added to 1 g of sample; this was then homogenised and left at room temperature (22 ± 1 °C) (to evaporate the methanol and dry the extract). Next, 5 mL of distilled water was added to 0.01 g of the dry extract, and this was shaken with a digital vortex (Thermo Scientific®) for 20 min. To 650 μL of this solution, 75 μL of 5% NaNO₂ was added, stirred and rested for 6 min. After this, 150 μL of 10% AlCl₃ was added, shaken and rested for 4 min. Finally, 500 μL of NaOH (1 M) and 1,150 μL of distilled water were added. The absorbance at 510 nm was recorded. A standard catechin curve (Sigma Aldrich) was constructed with concentrations of 0, 20, 40, 60, 80 and 100 mg · L⁻¹. The results are expressed in milligrams of quercetin equivalents (QE) per gram FW.

Superoxide dismutase (SOD; Enzyme Commission [EC] number: 1.15.1.1) activity was assayed by monitoring the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT), according to the method described by Giannopolitis and Ries (1977) and modified by Sánchez et al. (2005). Enzyme activity is reported as units per minute per gram FW, where 1 unit of SOD activity corresponds to the amount of enzyme required to cause a 50% inhibition of NBT reduction as evaluated at 560 nm. The extraction and analysis of total H₂O₂ used a colourimetric method (Sánchez et al., 2000). The results are reported in micromoles of H₂O₂ per minute per gram FW (total peroxides). Last, the extraction and determination of enzymatic activity of the enzymes catalase and guaiacol peroxidase were determined by the method described by Sánchez et al. (2000). The results are expressed as moles of H₂O₂ per minute per gram FW and nanomoles glutathione (GSH) per minute per gram FW, respectively.

Antioxidant capacity (AC) was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH˙) method. Aqueous ethanol (80%) was used for sample preparation. The DPPH’ assay was carried out in accordance with the procedure followed by Brand-Williams et al. (1995). Briefly, 0.3 mL of extract and 5.7 mL of DPPH were mixed at a concentration of 0.0375 g · L⁻¹. The mixture was kept in the dark for 30 min. The decrease in the quantity of DPPH radical was measured at 515 nm. The results are expressed as percentage inhibition of DPPH.

Prior to statistical analysis, the normal distribution of the data was confirmed by the Shapiro–Wilk test (p ≤ 0.05) (Hanusz and Tarasińska, 2015). Statistical analysis consisted of a general linear model with year and treatment effects (NSCs, yield, nut weight, kernel percentage and foliar nutrient concentration), and the remaining parameters were analysed only by treatment in the year 2020. The comparison of means was carried out with a multiple comparison of means with Tukey’s test (p ≤ 0.05). The data were analysed with the statistical package Statistical Analysis Software (SAS/STAT | SAS), version 9.3 (SAS Institute Inc., Cary, NC, USA).

It is well known that carbohydrates are key molecules in plants since they are part of the plant structure (e.g. cellulose) and are also the primary source of metabolic energy (e.g. starch) (Ojeda-Barrios et al., 2022). Here, the leaflets sprayed with sulphates, chelates and zinc NPs presented only slight numerical differences in the concentrations of NSCs (fructose, glucose, sucrose and starch), so it was not possible to identify a significant effect on NSCs between the two growing seasons examined (Table 1). These results are important, considering the negative impact of alternate bearing (AB) on pecan nut yield and quality (Wood et al., 2003; Rohla et al., 2007). Pecan trees quite commonly present AB; while the intensity of AB can be linked to the concentration of carbohydrates, it can also involve elements of agronomic management, such as irrigation, fertilisation, pruning and crop load. Other more random factors, such as water stress, lack of winter cold, tree age and cultivar, can also be involved (Wood et al., 2003). In Mexico, the main cultivars in commercial production are ‘Western Schley’, ‘Wichita’ and, more recently, ‘Pawnee’. Previous studies carried out to evaluate the intensity of AB in young and adult pecan trees by Conner and Worley (2000) report intermediate values in ‘Wichita’ (0.51 and 0.67) and ‘Western Schley’ (0.65 and 0.68); however, in ‘Pawnee’, there are no reports of AB, so it can be inferred that this is a promising new cultivar for pecan production.

The dormancy processes (paradormancy, endodormancy and ecodormancy) in temperate fruit trees are linked to various governing influences, including the environment and the genotype (Pertille et al., 2021). Among the internal factors governing dormancy are the concentrations of carbohydrates in leaves and buds (Wood et al., 2003; Rohla et al., 2007). In this study, foliar applications of Zn (NZN, ZnSO₄, Zn-EDTA and ZnO NPs) did not affect (p ≥ 0.05) the concentration of NSCs in the leaflets (Figure 3). Thus, in pecan trees, after nitrogen (N), Zn is the most important nutrient in terms of the number of fertiliser applications required and economic benefit generated by this activity (Heerema et al., 2017). However, for pecan, there is no information on the relationship between foliar applications of Zn and the NSC concentration in the leaflets. It is well known that, for pecan, flower development depends on reserve carbohydrates and the concentration of nutrients, including that of Zn (Tsuchida et al., 2015; Breen et al., 2018).

Figure 3.

On the other hand, we did find variation (p ≤ 0.05) between years with respect to yield and nut weight (grams per kilogram), but not for kernel percentage (Table 2). This behaviour indicates the occurrence of AB and the null effect of Zn applications. However, the kernel percentage was maintained across the two seasons, which is an aspect of high economic importance to farmers and is highly appreciated by marketers and consumers of this nut. The values of these parameters are similar to those reported by Castillo-González et al. (2019) in young trees (≈12 years old) of ‘Western Schley’ grown under Zn-sufficiency conditions. According to the NMX-FF-084-SCFI-2009 (NOM-2009) for improved nuts, with the exception of treatment with ZnSO₄ (Figure 4), the nuts harvested in this experiment correspond to Quality II, i.e. 140–170 nuts · kg⁻¹ and 58% kernel weight.

Figure 4.

The data generated with the Zn spray and the plants’ responses to the foliar nutrient application between growing seasons are shown in Table 3. Previous studies have shown the nutritional effects of N and Zn²⁺ on the establishment and commercial production of pecan nuts (Cruz-Álvarez et al., 2020; Smith et al., 2021). Nitrogen is an essential nutrient in the accumulation of biomass, including for the development of shoots and leaflets. Its major roles in trees include its association with chlorophyll biosynthesis and the production of sugars by photosynthesis (Moran-Duran et al., 2020). Similar foliar N concentrations are reported by Pond et al. (2006) when determining the levels of foliar nutrients in ‘Western Schley’ pecan from high-yield commercial orchards established in the USA (Arizona, New Mexico and Georgia) (25.5, 24.7 and 27.2 g · kg⁻¹) and in Sonora (Mexico) (24.8 g · kg⁻¹). The variations between the values are probably associated with the physicochemical properties of the soil and the agronomic managements between growing seasons. On the other hand, in our study, the Zn concentrations fluctuated between 35.81 mg · kg⁻¹ and 48.46 mg · kg⁻¹, wherein NZN and ZnSO₄ showed the best results. These results can be considered within the sufficiency range (30–100 mg · kg⁻¹), as reported by Walworth and Heerema (2019). In contrast, Pond et al. (2006) report a foliar Zn sufficiency concentration of 54.6 mg · kg⁻¹; however, our trees did not show visible deficiency symptoms (internode shortening, chlorosis and interveinal necrosis).

In most established pecan orchards, soil P is a relatively immobile element, and banding has been used to rapidly ameliorate its deficiency (Smith, 2009). In our study, the Zn spray did not maintain the concentration of P in the leaflets between the years evaluated. Similar results were reported by Naira et al. (2013) with foliar applications of 0.5% Zn (ZnSO₄), urea + ZnSO₄ (0.5%+0.5%), boric acid + ZnSO₄ (0.1% + 0.5%) and the combination of these in ‘Western Schley’ nuts, with values of 1.6, 1.8 and 2.2 mg · kg⁻¹, respectively. In pecan nuts, the information generated for this element with foliar applications of Zn is scarce. However, Zn remained at sufficiency levels for pecan, according to Ojeda-Barrios et al. (2012), with values between 1.05 mg · kg⁻¹ and 1.67 mg · kg⁻¹. On the other hand, with the exception of treatment with Zn-EDTA, no changes in K⁺ levels were observed. However, in this experiment, no significant changes were observed in kernel percentage, a characteristic associated with the optimal supply of Zn in pecan nut. The results are consistent with those published for ‘Western Schley’ (Ojeda-Barrios et al., 2014) with foliar application of Zn-EDTA (0.76, 1.53 and 2.29 mM). In contrast, lower concentrations of 8.4, 9.1, 8.6 and 11.1 mg · kg⁻¹ were reported by Naira et al. (2013) after spraying with ZnSO₄, urea + ZnSO₄, boric acid + ZnSO₄ and ZnSO₄ + amino acids, respectively.

Over the 2 years evaluated, Zn application did not affect the concentrations of Ca²⁺ (30.4–33.7 mg · kg⁻¹) or Mg²⁺ (3.8–4.8 mg · kg⁻¹), which – according to Pond et al. (2006) – are in the high and normal ranges, respectively. The leaflets did not present any necrosis related to high calcium concentrations. Mg²⁺ deficiency has been reported in acid and alkaline soils (Sparks, 1996). The soil characteristics were as follows: pH value: 7.8 and CaCO₃ content: 20%. These can interfere with the high concentration of Ca in the soil and can also affect Mg, as indicated by Walworth and Heerema (2019). Among the micronutrients, the concentrations of Fe²⁺, Cu²⁺ or Mn²⁺ were not affected by Zn-EDTA; however, the levels of Cu and Mn were similar to those observed with ZnSO₄. Similar behaviour was observed for Cu and Mn in a previous study carried out by Ojeda-Barrios et al. (2014) with Zn-EDTA sprays (0.76, 1.53 and 2.29 mM) over 3 years with young trees (≈8 years old) of ‘Western Schley’.

In general, Zn applications improve nut yield in pecan, but they have also been shown to increase the occurrence of AB (Castillo-González et al., 2019). In our study, Zn applications (NZN, ZnSO₄, Zn-EDTA and ZnO NPs) maintained the concentrations of the essential elements with the exception of Zn²⁺, whereby they did not exceed the control values (Figures 5A and 5B). A similar behaviour was observed by Naira et al. (2013) with foliar ZnSO₄ sprays, with maximum values of Zn between 195 mg · kg⁻¹ and 294 mg · kg⁻¹, i.e. under toxic conditions according to Walworth and Heerema (2019). In this sense, cultivated plants can vary their nutrient composition and show optimum growth and development (Cruz-Crespo et al., 2014). Most pecan orchards are established in arid or semi-arid regions, and growers have managed Zn supply through multiple foliar Zn sprays, mainly ZnSO₄ and Zn-EDTA, with Zn-EDTA proving more efficient in alkaline soils (Walworth et al., 2017). Other authors such as Smith and Storey (1979) point out that Zn(NO₃)₂ sprays show better Zn absorption than ZnSO₄ for similar doses. In general, the other Zn sources, including NPs, show a similar behaviour. Results can be associated with the relatively high dose and number of applications made. However, foliar application of ZnO NPs at concentrations between 0.1 mg · L⁻¹ and 150 mg · L⁻¹ have been shown to improve the nutritional status of numerous crops including mango (M. indica L.) (Elsheery et al., 2020), pomegranate (P. granatum L.) (Davarpanah et al., 2016), coffee (Coffea arabica L.) (Rossi et al., 2019) and common bean (P. vulgaris L.) (Bautista-Díaz et al., 2021).

Figure 5.

Plants under abiotic stress activate a defence system that includes antioxidant enzymes (peroxidases, SOD, catalase and ascorbate peroxidase) and antioxidant compounds (phenols, flavonoids, GSH and carotenoids, among others), which can inhibit or reduce the production and effects of reactive oxygen species (ROSs) (Teran-Erazo et al., 2019). These ROSs are associated with oxidative damage and cell death, and in pecan nut trees, they can influence AB by reducing growth and productivity (Ojeda-Barrios et al., 2022). Our study explored the behaviour of some antioxidant enzymes in the leaflets, including SOD, CAT and guaicol peroxidase (GPx) (Table 4). Zinc is a micronutrient that has stabilising properties and protective effects against oxidative and peroxidative damage, loss of integrity and alteration of membrane permeability (Subba et al., 2014). In general, Zn-EDTA spraying significantly reduced the activities of SOD, CAT and H₂O₂ production. However, ZnSO₄ and ZnO NPs showed similar values for CAT and H₂O₂. These results are likely linked to the activity of Zn as a cofactor for several enzymes, including dehydrogenases, oxidases and peroxidases (Balafrej et al., 2020).

Applications of200 mg · L⁻¹ Zn increased the activity of GPx (5.19-5.64 nmol GSH · min⁻¹ · g⁻¹), which is associated with a reduction in peroxide production. An increase in GPx activity has been reported with edaphic applications of Zn(NO₃)₂·6H₂O (150, 300 and 600 μM) in Chenopodium murale L. (Zoufan et al., 2018). The role of Zn in the peroxidation of cell membrane lipids and the increase in activity of several antioxidant enzymes caused by generation of O^-₂ and H₂O₂, including GPx, have been reported (García-López et al., 2019). Similarly, this behaviour may be caused by the maintenance of the balance in the O^-₂ and H₂O₂ contents under Zn-stress conditions (Ibrahim and Ramadan, 2015).

Under abiotic stress, plant cells can increase their AC to minimise the negative effects of oxidative stress on physiological and biochemical processes (Davarpanah et al., 2016). In our study, the values of AC in the leaflets fluctuated between 76.9% and 83.1% of DPPH inhibition, the application of Zn-EDTA standing out among the treatments (Table 4). None of our trees showed symptoms of Zn deficiency, as was also corroborated by the sufficiency range measurements of their foliar Zn. However, in pecan tree, there is little information on Zn spraying and changes in the AC. Subba et al. (2014) indicate that Zn plays important roles in non-enzymatic antioxidant systems and is a micronutrient that helps stabilise the effects of oxidative and peroxidative stress, loss of cell integrity and permeability.

One of the most reliable indicators of stress in plants is associated with the synthesis and accumulation of photosynthetic pigments, where Zn actively participates in their biosynthesis (Subba et al., 2014). The concentrations of the photosynthetic pigments, chlorophyll and carotenoids, are presented in Table 5. With Zn-EDTA, decreased concentration of chlorophyll and total carotenoids was determined, while with ZnSO₄ and ZnO NPs, the concentration of chlorophyll and total carotenoids was reduced. Foliar Zn uptake depends on the Zn source. ZnSO₄ and Zn(NO₃)₂ are most common in pecan. Most of the studies on foliar application of ZnO NPs are for vegetables and cereals; there is little information for perennial plants, such as vines and fruit trees, especially not for pecan trees. Previous studies were conducted on the spraying of ZnO NPs as follows: 150 mg · L⁻¹ in the common bean (P. vulgaris L.) (Bautista-Díaz et al., 2021); 50 mg · L⁻¹ in broad bean (Vicia faba L.) (Ghidan et al., 2020); and 0.10 mg · mL⁻¹ in carrot (Daucus carota L.) (Siddiqui et al., 2019). These experiments showed significant effects on the concentrations of chlorophyll and carotenoids.

Among the bioactive compounds that we evaluated in pecan leaflets, TP and TFl did not show significant variation due to Zn application (Table 5). This lack of response may be associated with the metabolic role of Zn, including synthesis of proteins, regulation of carbohydrate metabolism and the activation of some antioxidant enzymes that act against oxidative and peroxidative cell damage (Elsheery et al., 2020). In potato leaves (Solanum tuberosum L.), for 0.4% ZnSO₄ sprays before and after flowering, Korkmaz et al. (2018) found that there was a direct relationship between TP concentration and the increase in the Zn dose, but there was no effect of application time.