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Article

Seed-Primed and Foliar Oxozinc Nanofiber Application Increased Wheat Production and Zn Biofortification in Calcareous-Alkaline Soil

by
Wiqar Ahmad
1,2,3,†,
Zhiyou Zou
1,*,†,
Muhammad Awais
2,
Fazal Munsif
4,
Aziz Khan
5,
Jaya Nepal
6,
Masood Ahmad
7,
Sultan Akbar
8,
Ijaz Ahmad
9,
Muhammad Shahid Khan
10,
Zahid Qamar
11 and
Haroon Khan
12
1
School of Earth Sciences, Yangtze University, Jingzhou 434025, China
2
Department of Soil and Environmental Sciences, The University of Agriculture Peshawar, Amir Muhammad Khan Campus Mardan, Mardan 23200, Pakistan
3
Soil and Water Science Department, Indian River Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, 2199 South Rock Road, Fort Pierce, FL 34945, USA
4
Department of Agronomy, The University of Agriculture Peshawar, Amir Muhammad Khan Campus Mardan, Mardan 23200, Pakistan
5
Key Laboratory of Grassland Agroecosystems, Lanzhou University, Lanzhou 730000, China
6
Department of Soil, Water and Ecosystem Sciences, Indian River Research and Education Center, University of Florida, Fort Pierce, FL 34945, USA
7
Department of Horticulture, The University of Agriculture, Peshawar 25000, Pakistan
8
Department of Plant Breeding and Genetics, The University of Agriculture, Peshawar 25000, Pakistan
9
Department of PBG, The University of Agriculture Peshawar, Amir Muhammad Khan Campus Mardan, Mardan 23200, Pakistan
10
Department of Plant Protection, The University of Agriculture, Amir Muhammad Khan Campus, Peshawar 25000, Pakistan
11
Department of Applied Physics, Federal Urdu University of Science and Technology (FUUAST), Islamabad 44000, Pakistan
12
Department of Weed Science & Botany, The University of Agriculture, Peshawar 25000, Pakistan
 
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*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2023, 13(2), 400; https://doi.org/10.3390/agronomy13020400
Submission received: 3 January 2023 / Revised: 24 January 2023 / Accepted: 25 January 2023 / Published: 30 January 2023
(This article belongs to the Special Issue Physiology of Abiotic Stress Tolerance in Crops at Reproductive Stages and Crop Improvement Strategies)
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Abstract

:
Low Zinc (Zn) availability in alkaline calcareous soil is one of the major causes of low cereal yield and quality. Conventional application of Zn sulfate (ZnSO4) fertilizer through soil application attains minimal Zn efficiency as it is readily fixed in such soils. Oxozinc nanofiber (ZnONF) was evaluated for wheat Zn biofortification using different application methods to tackle this issue. Pots in triplicate (each with 7 kg soil) were arranged in a completely randomized design with a control treatment without Zn application. The conventional ZnSO4 fertilizer recommended dose (5.5 µg Zn kg−1 of soil) was used for comparison and applied through soil addition, foliar spray, and seed priming, while the ZnONF was applied through foliar spray, seed coating, and seed priming (@ 0.5 kg ha−1) either alone or in combination with ½ZnSO4 applied to the soil. The application of ZnONF significantly improved wheat plant growth as evidenced by increased plant height (14.5%), spikelets per spike (13.7%), and Zn use efficacy (611%) regardless of application methods as compared to control. The highest Zn uptake efficiency (34%) for nanofibers was obtained for theseed primed, followed by seed coating (23%) and foiar application (7%), respectively. Moreover, at the combined ZnONF and ½ZnSO4 application, further improvements for spike length, number of spikelets spike−1, grain, leaf, root, and stem Zn concentrations, as well as their respective Zn contents, were noted. These results elucidated that Zn nutrition with ZnONF was either at par with or higher than the conventional ZnSO4 fertilizer application despite significantly reduced ZnONF quantity, irrespective of the application method used. Additionally, the combined ZnONF and ½ZnSO4 (foliar spray, seed coating, or seed priming) maximized the crop Zn accumulation, wherein the ½ZnSO4 + ZnONF through foliar application exceeded grain Zn biofortification. Thus, various Oxozinc nanofibers application modes may be recommended for wheat biofortification either separately or in combination with ZnSO4 in Zn deficient calcareous soils for improved Zn nourishment.

1. Introduction

Alkaline calcareous soils with continuous cereals cultivation have decreased crop productivity and quality due to declining soil fertility in developing countries [1]. Among essential plant nutrients, Zn is one widely deficient micronutrient in cereal-based cropping system areas [2]. Low soil availability is a major hurdle to achieving high crop production [3]. Zn deficiency is most common in alkaline calcareous soils having low phyto-available Zn concentration [4,5], comprising around 50% of the agricultural land in the world [3]. Resultantly, low Zn crops produced on such soils do not meet human bodily and functional requirements, which results in various health issues [6]. Nutritional deficiency reduces production and adversely affects the crop quality required for proper human nutrition; therefore, improving grain Zn content would reduce the intensity of Zn deficiency-associated health problems in humans. The World Health Organization’s recommended daily intake of Zn for an adult human is 15 mg day−1; however, around 25% of the world’s population, mostly in developing countries, is less than that amount. However, Zn deficiency could be reduced in plants by following various practices, i.e., supplementary application, using diverse diets, and food and crop plants bio-fortification [6]. Conventionally, mineral ZnSO4 fertilizers are most commonly used because of their high solubility and low price [7]. However, fixation reactions reduce zinc bioavailability in soils with low organic matter, high carbonate content, and high pH, causing calcite adsorption or Zn(OH)2 or ZnCO3 precipitation, making conventional fertilizers ineffective for crop zinc uptake [5,8,9]. Wheat is one of the major staple foods for approximately 40% of the world’s population living in developing countries [10]. Zn biofortification of wheat grain is a technique involving improving its inherent Zn status through the external application of Zn in the form of solid or liquid fertilizers to the crop at the proper growth stage. With the global population expected to reach 9.8 billion by 2050 [11], a major challenge for scientists is not only to increase food production by multiple folds but also to enhance the nutritional qualities of the food produced to feed more people, particularly in developing regions [12]. This would require not only improving the current technology but also the identification of new areas of research in this field. One such promising technology is nanotechnology, i.e., the use of engineered nanomaterials that are a billionth of the size of a meter and offer unique properties owing to their minute dimensions.
Recently, nanotechnology has been getting attention for mitigating nutritional stress and securing sustainable crop management required for potential production. Given their small size, large specific surface area, and high reactivity, nanoparticles (NPs) have great application potential in agriculture [13]. Nanoparticles of essential elements may serve as essential plant nutrients [14,15,16,17]. Supplementing Zn via nano Zn alternatives offer a potential in wheat Zn biofortification, especially in alkaline calcareous soils where Zn is easily fixed in soils. Zn oxide (ZnO) nanoparticles have been widely tested [14] for crop growth, showing effectiveness at low (≤100 mg kg−1) concentrations. In contrast, at a high concentration (1000 mg kg−1), ZnO nanoparticles enhanced cucumber crop Zn accumulation but not crop growth [18]. Nanoscale powders of different elements can be used as fertilizers and pesticides [19] with efficient and controlled release of pesticides, herbicides, and fertilizers and the detection of soil moisture and nutrients. Nano-fertilizers are readily absorbable by plants and possess the potential to increase growth and yield [20]. Nano ZnO particles with a size range of 25 nm at 1000 ppm concentration demonstrated improved germination, seedling vigor, root and stem growth, chlorophyll content, and pod yield by 34% in peanuts [21]. ZnO nanoparticle colloidal solution is used as fertilizer. It serves as a plant’s nutrient source and prevents the use of synthetic fertilizers, thus reviving the soil to an organic state [20]. Nano-fertilizers are used in minimal quantity compared to conventional fertilizers yet have been proven to increase wheat yield by 20–25% [22].
ZnO nanoparticles for Zn deficiency mitigation and crop biofortification have been widely reported. However, the application of ZnO nanofibers for crops’ Zn biofortification has rarely been studied. Through the well-known laboratory procedure of electrospinning, nanofibers can be prepared from zinc nanoparticles to enhance their qualities. Nanofibers of desired size and shapes can be produced from polymeric solutions using electrospinning techniques under controlled laboratory conditions [23]. The Zn nanoparticles can be fabricated into specific nanofibers to achieve high surface area, porosity, and reactivity, which can potentially improve crop uptake.
The main objective of this study was to evaluate the effectiveness of novel Oxozinc (ZnO) nanofibers as a Zn nano-fertilizer applied through priming or foliar in comparison with conventionally used ZnSO4 under alkaline calcareous silty loam soil conditions. We hypothesized that because of the higher surface area and reactivity of Zn nanofibers compared to conventional fertilizers, Zn use efficiency and wheat crop quality would be improved at a lower application amount.

2. Materials and Methods

2.1. Preparation and Characterization of ZnO Nanofiber

Oxozinc nanofiber (ZnONF) was prepared locally in the laboratory using the electrospinning method following the protocols described by [24]. In brief, ZnO nanoparticles were first prepared using the chemical precipitation method from Zn acetate (C4H6O4Zn) and sodium hydroxide (NaOH) reaction, as described by [25]. The Zn nanoparticle obtained was then mixed with polyvinyl alcohol (PVA) at a ratio of 1:2, and the solution was mounted on the electrospinning injector with a needle hole diameter of 1 mm. The injector was set at 0.5 mL/h outflows in front of an aluminum foil at a 20 cm distance, receiving the solution jet with nanofibers. The prepared ZnO nanofibers were characterized using a scanning electron microscope (SEM; Model: JSM-5910; Make: JEOL, Japan; Energy: 30 KV Magnification) (Figure 1) and X-Ray Diffractometer (XRD; Model: JDX-3532; Make: JEOL, Japan; Voltage: 20–40 kV; Current: 2.5–30 mA; X-Rays: CuKa (Wavelength = 1.5418 Å); 2Theta-Range: 0 to 160°) (Figure 2) for the material identification. It was observed using SEM (Figure 2) that nanoparticles that were subjected to electrospinning were round, irregular, and hexagonal in shape and individual as well as compounded. The pure nanofibers selected for the experiment were chosen from the batch, as observed in Figure 2c,d. The XRD spectra (Figure 2) confirmed the formation of nanofiber and revealed that peaks existed at 2 θ between 30 and 40 [26].

2.2. Application of ZnO Nanofibers to Wheat Crop in Pots

The study was conducted in pots (8″ top, 6″ bottom, and 12″ height accommodating 7 kg soil) with 10 treatments and 3 replications arranged in a completely randomized design. The experimental treatments include Control, ZnSO4(f), ZnSO4(s), ZnSO4(p), ZnONF(f), ZnONF(c), ZnONF(p), ½ZnSO4(s) + ZnONF(f), ½ZnSO4(s) + ZnONF(c), ½ZnSO4(s) + ZnONF(p) where the suffix f, s, p, and c means application through foliar, soil addition, seed priming, and seed coating, respectively. Soil for pots was collected from a cultivated field (Table 1). The concentration of ZnSO4 was 0.2% for foliar application and seed priming and 5.5 µg kg−1 of soil for soil application as per local recommendations. The concentration of ZnO nanofiber was 0.016% for foliar and 0.05% for seed priming application and seed coating using polyvinyl alcohol (PVA) as a coating agent and an electrospinning machine as a coating instrument. The lower application rate for Zn oxide nanofiber was selected based on seed germination tests and preliminary trials in the laboratory. For seed coating, healthy seeds were selected and placed in aluminum foil to receive the nanofiber solution spray on the seeds. Seeds inside the aluminum foil were shuffled at a constant interval for uniform coating results [27]. Wheat (Triticum aestivum L.) seed, variety Wadan-2017, purchased from the Cereal Crop Research Institute Pir Sabak, Nowshehra, Khyber Pakhtunkhwa, Pakistan, was sown at the rate of 9 seeds pot−1. Plants in pots were then thinned to 4 pot−1 until maturity. The crop was harvested 160 days after sowing (DAS). A filtrate collected from a 1:5 soil:water suspension was sent to an EC meter (DDS-11A, Nanjing, China) for electrical conductivity measurement [28], and a pH meter (HM-12P, Japan) (1:5 H2O) was used to determine soil pH. A hydrometer procedure was used to determine soil texture [29]. Soil organic matter was determined using the chromic acid wet oxidation method [30]. Soil-available Zn, P, and K were extracted from 1:2 soil:ammonium bicarbonate di-ethylene tri-amine penta-acetic acid (AB-DTPA) with shaking for 30 min and filtration with Whatman 42 filter paper [31]. Soil-available Zn was determined using an atomic absorption spectrophotometer (Perkin Elmer Model 2380, Champaign, IL, USA) directly in the filtrate. Available P was determined through color development with ascorbic acid and reading with a spectrophotometer (U-3900H, Hitachi-Hitech) at 880 nm. Available K was determined with a flame photometer (Jenway-PFP7). The pictorial view of the experiment at different stages is given in Figure 3.

2.3. Data Collection

In each pot, the plant’s height was measured with measuring tape stretched from the bottom to the top of the plant. Spike length was also measured with a measuring tape, and the number of spikes spike−1 was counted manually in each pot. At maturity, spikes in each pot were counted and threshed to calculate the number of grains spike−1.

2.4. Plant Sample Analysis

Zn concentration in straw, grain, leaves, and root samples was determined through wet acid digestion (HNO3/HClO4 digestion) [32] and subsequently analyzed for Zn concentration on an atomic absorption spectrophotometer. Zn uptake (µg pot−1) was calculated as
Zn uptake = (Straw yield × Zn concentration (mg kg−1) + (Grain yield × Zn concentration) + (leaves dry weight × Zn concentration) + (root dry weight × Zn concentration)
Zn uptake efficiency (ZUE) was determined as
Z U E   ( % ) = Z n   c o n t e n t t r e a t m e n t − Z n   c o n t e n t c o n t r o l Z n   a p p l i e d   × 100

2.5. Statistical Analysis

ZnO nanofiber and ZnSO4 application and replications were treated as fixed and random effects, respectively. Normality and homoscedasticity assumptions of the parametric test of all studied traits were checked prior to statistical analysis using the Shapiro–Wilks and Levene’s test, and it was found that collected data were normally distributed. After verifying the normality of the data, they were analyzed and presented using analysis of variance (ANOVA). The experimental data on the investigated parameters were subjected to a variance analysis suitable for CRD design using StatistiX 8.1 computer software. For parameters with significant F-values (p ≤ 0.05), a post-hoc analysis using the LSD test was performed to assess significant differences between the means. At a significance threshold of p ≤ 0.05, the Least Significant Difference (LSD) test was employed. [33].

3. Results

3.1. Growth Parameters

The effects of different treatments on wheat growth and yield parameters (plant height, spike length, number of spikelets per spike, and grains per spike) are presented in Table 2. Use of ZnSO4 fertilizers and ZnO nanofibers (ZnONF) through either mode of application to the wheat crop showed significant (p ≤ 0.05) improvement in plant height and non-significant improvement (up to 1.6 cm) in spike length (Table 2). Soil application, foliar spray, and seed priming of Zn through ZnSO4 fertilizer at the recommended dose (5.5 µg kg−1 of soil) recorded 92, 86, and 88 cm plants with 18, 11, and 13% increase in plant height over the Zn control, respectively. However, the ZnO nanofiber seed priming (ZnONF(p)), seed coating (ZnONF(c)), and foliar application (ZnONF(f)) showed 94, 92, and 82 cm plants with 20, 18, and 6% increase in plant height over the Zn control, respectively. Combined ½ZnSO4(s) (half of the recommended ZnSO4 applied to soil) in combination with either of the ZnONF seed coating (½ZnSO4(s) + ZnONF(c)), seed priming (½ZnSO4(s) + ZnONF(p)), and foliar application (½ZnSO4(s) + ZnONF(f)) also showed significantly (p ≤ 0.05) improved plant height (92, 92, and 85 cm, respectively) with 18, 18, and 9% increase over the Zn control, respectively. Spike length with ZnSO4 priming, foliar, and soil application was 11.7, 11.3, and 11.2 cm, respectively. Using ZnO nanofiber applications as seed coating, priming, and foliar, the spike length was 12.0, 11.8, and 11.0 cm, respectively, while these modes of ZnONF when combined with ½ZnSO4 applied to soil, spike length was 12.1, 11.2, and 11.3 cm, respectively compared to spike length in the control (10.5 cm).

3.2. Yield Parameters

Application of ZnSO4 fertilizers or ZnONF by either method to wheat crop significantly (p ≤ 0.05) enhanced the spikelets and grain count spike−1 (Table 2). The spikelets count spike−1 and grains count spike−1 with ZnSO4 applied to soil (ZnSO4(s)) at 5.5 µg kg−1 of soil were 19.3 and 51. For ZnSO4 treated as a foliar spray (ZnSO4(f)), the spikelets count spike−1 and grain count spike−1 were 18.7 and 47, while for ZnSO4 seed priming (ZnSO4(p)), these were 19.3 and 44.3, respectively. The increase in spikelet count spike−1 with ZnSO4 application as soil, foliar, and seed priming was 9, 6, and 9%, and grain count spike−1 was 30, 20, and 11% over the control, respectively (Table 2). The ZnONF coating, priming, and foliar application recorded spikelets count spike−1 of 20.7, 20.7, and 19, showing an edge of 17, 17, and 7%, and grain count spike−1 was 52.3, 52.0, and 43.7 with 33, 32, and 11% increase over the control, respectively. A combination of ½ZnSO4 applied as soil with ZnONF each as foliar spray (ZnONF(f)), seed coating (ZnONF(f)), and seed priming (ZnONF(f)) registered the spikelets count spike−1 of 20.7, 20, and 18.7, with a 17, 13, and 6% increase over the control (17.7), respectively, and grains count spike−1 of 49, 42, 50.3 with a 25, 7, 28% increase over the control (39.3), respectively.

3.3. Zn Concentration in Plant Tissues

ZnSO4 fertilizers and ZnONF treatments through different modes of application to wheat crops significantly affected the Zn concentration in grain, leaves, stem, and roots (p ≤ 0.05; Table 3). Wheat grain exhibited maximum Zn concentration (29 µg g−1) with ZnSO4 foliar spray at the recommended dose (5.5 µg kg−1 of soil); however, when ZnSO4 was treated as seed priming or applied to the soil at the recommended dose, Zn concentration in grain was 26 and 25.6 µg g−1, respectively. While all these values were statistically similar, their respective increase in grain Zn concentration over the control was 39, 25, and 23%. When ZnONF as foliar spray, seed coating, and seed priming was applied, the grain Zn concentration was 29.3, 26.1, and 25.7 µg g−1, achieving an increase in grain Zn concentration by 41, 25, and 24%, respectively, over the control. The combined application of ½ZnSO4 as soil and ZnONF as foliar spray and seed priming resulted in significantly (p ≤ 0.05) higher grain Zn concentration (32.9 and 31.1 µg g−1, respectively) over all the other treatments. Combined ½ZnSO4 as soil and ZnONF applied as seed coating recorded significantly (p ≤ 0.05) lower grain Zn concentration (25.7 µg g−1) as compared to other Zn treatments with a similar mode of application (foliar spray and seed priming), whereas each one registered an increase of 58, 49, and 23%, respectively, over the control.
In leaves, Zn concentration (21.2 µg g−1) was the maximum for the recommended dose (5.5 µg kg−1 of soil) of ZnSO4 through foliar application, while for soil application and seed priming, it was 20.8 and 18.8 µg g−1, respectively, where each one achieved an increase of 19, 17, and 6% over the control (17.8 µg g−1), respectively. For ZnONF, the maximum leaf Zn concentration was noted with ZnONF seed coating (20 µg g−1), followed by ZnONF seed priming (19.1 µg g−1) and ZnONF foliar spray (18.1 µg g−1), where each one registered an increase in Zn content in leaf by 13, 7, and 6%, respectively, over the control. In the combined ½ZnSO4 applied as soil and ZnONF applied each as seed priming, seed coating, and foliar spray, the leaf Zn concentration was 23.3, 22.4, and 18.9 µg g−1, where each one registered an increase of 31, 26, and 6% over the control, respectively.
The root Zn concentrations for ZnSO4 as foliar spray, seed priming, and soil addition at the recommended dose (5.5 µg kg−1 of soil) were 53.5, 36, and 26.3 µg g−1, and recorded an increase of 149, 68, and 23%, over the control (21.47 µg g−1), respectively. For ZnONF applied through seed coating, foliar spry, and seed priming, the root Zn concentrations were 37.4, 34.1, and 29.3 µg g−1, each one accruing an increase of 74, 59, and 36%, respectively, over the Zn control. In the case of ½ZnSO4 applied as soil combined with ZnONF applied as either seed priming, foliar spray, or seed coating, the root Zn concentrations were 40.8, 38.4 µg g−1, and 28 µg g−1, where each one registered an increase of 90, 79, and 30%, respectively, over the control. With regards to stem Zn concentration, application of ZnSO4 foliar spray, seed priming, and soil addition at the recommended dose (5.5 µg kg−1 of soil) recorded 17.5, 16.6, and 15 µg g−1 stem Zn concentration while having a 58, 49, and 35% increase over the control (11.1 µg g−1), respectively (Table 3). For ZnONF seed priming, seed coating, and foliar spray, the stem Zn concentrations were 20.4, 16.6, and 11.7 µg g−1, showing 84, 50, and 5% higher stem Zn concentrations over the control, respectively. However, the ½ ZnSO4 applied as soil combined with ZnONF either as seed priming, seed coating, and foliar spray recorded 20.2, 16.5, and 12.9 µg g−1 stem Zn concentration, each one registered an increase of 82, 48, and 16%, respectively, over the control.
Table 3 shows that Zn fortification through seed priming of the wheat crop with ZnONF resulted in the maximum and significantly higher Zn uptake efficiency (ZUE, 34%) than the rest of the treatments. Application of ZnONF to the wheat crop as seed coating with a ZUE value of 23% followed the ZnONF priming, while the difference between the two higher ZUE values was significant. Application of the sole ZnSO4 to the soil through seed priming or foliar spray at the recommended level or its application in half of the recommended dose to soil plus ZnONF with either method (foliar, seed coating, or priming) did not reflect an increase or improvement in ZUE in wheat crop, while all of them were statistically similar. The ZUE for ZnSO4 applied to the soil at the recommended dose was 3%, while for foliar spray and seed priming, the ZUE was 2% each. When ½ZnSO4 applied to soil was combined with ZnONF as foliar spray, seed coating, and seed priming, the ZUE improved to 5, 4, and 6%, respectively; however, the improvement was still non-significant statistically and remained at par with ZnSO4 applied at the recommended dose to soil.

3.4. Zn Uptake

Application of ZnSO4 fertilizers and ZnONF through different methods to wheat crops significantly (p ≤ 0.05) affected the Zn accumulation in grain, leaf, root, stem, and total Zn uptake (Table 4). The grain total Zn content with ZnSO4 application as foliar spray, soil addition, and seed priming was 242, 233, and 211 µg pot−1, each one accruing an increase of 63, 58, and 43%, respectively, over the control (148 µg pot−1). The order of grain Zn content with the application of ZnONF through different methods was: seed priming (283 µg pot−1) > seed coating (232 µg pot−1) > foliar spray (215 µg pot−1) while each one accrued 92, 57, and 45% increase over the control, respectively. When ½ZnSO4 soil addition was combined with ZnONF application as foliar spray, seed priming, and seed coating, their respective grain Zn content (380, 334, and 252 µg pot−1) increased by 157, 126, and 71%, respectively, over the control.
The leaf total Zn content for ZnSO4 foliar spray was 127 µg pot−1, while for soil addition and seed priming, leaf Zn content was 114 and 105 µg pot−1, showing an increase of 51, 35, and 25% over the leaf Zn content in the control treatment (84 µg pot−1), respectively. With the application of ZnONF through seed coating, the leaf Zn content was 123 µg pot−1; with ZnONF application as seed priming, the leaf Zn content was 111 µg pot−1, while for its foliar spray, the leaf Zn content was 84 µg pot−1. Variation in leaf Zn content with ZnONF application as seed coating and seed priming showed an increase of 47% and 33%, respectively, over the Zn control, while ZnONF foliar spray recorded no change in leaf Zn content as compared with the control treatment. With ½ZnSO4 as soil addition in combination with ZnONF application either as seed coating, seed priming, or foliar spray, the leaf Zn contents recorded were 145, 139, and 129 µg pot−1 and the accrued increase in leaf Zn content by each treatment was 73, 66, and 53% over the Zn content in leaf recorded for the control treatment (84 µg pot−1).
In roots, Zn content with ZnSO4 treatment as foliar spray, seed priming, or soil addition at the recommended dose (5.5 µg kg−1 of soil) was 188, 103, and 76 µg pot−1, showing an increase of 149%, 36%, and 1% over the root Zn content in the control (75 µg pot−1) treatment, respectively (Table 4). Significant (p ≤ 0.05) variation in root Zn content was also observed with ZnONF application with seed coating, having root Zn content of 132 µg pot−1, seed priming with 113 µg pot−1, and foliar spray with 91 µg pot−1, where the increase over the control for each ZnONF application method was 75%, 49%, and 21%, respectively. With the application of ½ZnSO4 as soil addition in combination with ZnONF as seed priming, the root Zn content was 162 µg pot−1, followed by ½ZnSO4 as soil addition in combination with ZnONF foliar spray with root Zn content of 139 µg pot−1, and ½ZnSO4 as soil addition in combination with ZnONF seed coating with root Zn content of 87.0 µg pot−1, respectively, accruing an increase of 115%, 85%, and 15%, over the root Zn content in the control (75.4 µg pot−1), respectively.
Stem Zn content with the application of ZnSO4 as seed priming was 224 µg pot−1; with the application of ZnSO4 as foliar spray or soil addition, the stem Zn content was 220 and 211 µg pot−1, respectively, whereas each of the above application methods recorded an increase of 87%, 85%, and 77%, over the stem Zn content in the control pots (119 µg pot−1), respectively. Significant (p ≤ 0.05) variation in stem Zn content was observed for ZnONF application; as seed priming, the stem Zn content was 340 µg pot−1, followed by seed coating with stem Zn content of 235 µg pot−1, and then the foliar spray with stem Zn content of 126 µg pot−1, whereas the increase in stem Zn content for each ZnONF application method was 185%, 97%, and 5%, respectively, over the stem Zn content in the control (Table 4). With ½ZnSO4 applied as soil combined with ZnONF as seed priming, the stem Zn content was 291 µg pot−1, followed by ½ZnSO4 applied as soil combined with ZnONF as seed coating (stem Zn content 247 µg pot−1), and ½ZnSO4 applied as soil combined with ZnONF as foliar spray (stem Zn content 190 µg pot−1), and each one showed an increase in stem Zn content by 143%, 107%, and 59%, respectively, over the control (119 µg pot−1).
Total Zn uptake by wheat crop significantly (p ≤ 0.05) varied with different modes of ZnSO4 application at the recommended dose (5.5 µg kg−1 of soil) (Table 4); the total Zn uptake for ZnSO4 foliar spray was 776 µg pot−1, for seed priming and soil addition, the Zn total uptake was 642 and 633 µg pot−1, respectively. These quantities of Zn total uptake by wheat crop accrued through different modes of ZnSO4 application recorded an increase of 82%, 50%, and 48% over the Zn total uptake in the control (427 µg pot−1), respectively. The Zn total uptake with ZnONF seed priming was 847 µg pot−1, followed by Zn total uptake, with ZnONF seed coating (722 µg pot−1) and foliar spray (516 µg pot−1), each accruing 98%, 69%, and 21%, respectively, over the Zn control. However, ½ZnSO4 applied as soil combined with ZnONF seed priming recorded the maximum Zn total uptake of 926 µg pot−1. The ½ZnSO4 applied as soil plus ZnONF as foliar, and ½ZnSO4 applied as soil plus ZnONF as seed coating resulted in 838 and 731 µg pot−1 total Zn uptake by wheat crop while showing a 117%, 96%, and 71% increase over the Zn total uptake in the control, respectively.

4. Discussion

4.1. Effect of Zn on Wheat Growth and Yield Parameters

Plant height was statistically similar among the Zn source and methods of application, but all the Zn treatments were significantly (p ≤ 0.05) higher over the Zn control regardless of application method (Table 2). This means that ZnO nanofiber (ZnONF) was an effective alternative Zn source despite its application at a highly reduced rate (0.5 kg Zn ha−1) as compared to conventional ZnSO4 fertilizers. The soil was not only Zn deficient but also alkaline in nature and low in soil organic matter content (Table 1), which could further cause inhibition in Zn+2 absorption by the plants. Soil pH is the primary soil factor affecting Zn distribution in soil, wherein it is more readily released when the soil pH is acidic and more readily adsorbed on soil matrix at higher pH, especially in cases when the soil OM content is low [34]. However, any improvement in growth and yield under such Zn deficiency and alkaline conditions may be related to external Zn application [35]. The results also elucidated that both the seed coating and seed priming of ZnONF edged over the soil application of ZnSO4 because of their reduced chances of Zn fixation from ZnONF and improved chances of Zn absorption by the plants [36]. Foliar applied Zn from either ZnSO4 or ZnONF, or their combination rendered improvement in plant height and was 7–9% lower; the number of spikelet spike−1 and the number of grains spike−1 were 3 and 10% lower for foliar ZnSO4 and 10–22% for foliar ZnONF than their other counterpart methods of application viz soil application, seed coating, seed priming, or their combinations (Table 2). Although previous authors showed a more prominent effect of foliar application of ZnO NP on plant growth than any other Zn source [37], here, the low performance in plant growth using foliar application could be attributed to its application at mid or latter growth stages rather than other modes of application where Zn availability is increased at the start of the crop growth. Additionally, the shape and size of nanomaterial are key in affecting the absorption through the leaves more readily [38], and thus, nanofibers may not be as readily available through foliar application compared to other Zn oxide forms. Keeping the Zn sources constant, timely availability of Zn from either source and by any mode improves the plant growth through enhancing the growth hormone Indole Acetic Acid [39], chlorophyll content [40], photosynthetic activity [41], and enzymes acid and alkaline phosphatase, phytase, and dehydrogenase activities [41] resulting in improved plant height, spike length, spikelets spike−1, and grains spike−1.
Contrary to foliar application, ZnSO4 seed priming was 3% higher in spikelets spike−1 but 9% lower in grain spike−1 (Table 2), indicating a maximum of seed primed with Zn exhaustion until grain development. Soil application of ZnSO4 and seed coating and priming of ZnONF were the highest in spikelets and grain counts spike−1 (Table 2), perhaps due to Zn availability throughout the growth period. Previous workers also reported higher grain production with foliar ZnO NP application [37,40]. However, this study revealed ZnONF and ZnSO4 foliar application as synonymous with its improved grain yield rather than vegetative growth, revealing its translocation to grain development better than any other mode of application. The pitfall of lower vegetative growth in the case of foliar application was masked by its combined application with ½ZnSO4 as soil addition by recording 4–11% more spikelets spike−1 and 18% more grain spike−1 than the other modes of application (Table 2). In this case, the soil-applied ZnSO4 supports the initial crop growth, and foliar application of ZnONF supports the grain development along with crop growth. Studies by [42] support Zn application for higher grain and biomass yield, and [43] observed the highest grain yield and grain NP and Zn uptake from the mixture of ZnSO4 and foliar ZnONP compared to sole ZnSO4 fertilizer, while [41] reported a significant increase in shoot and root lengths, root area, chlorophyll, leaf protein, and biomass yield as a result of increased dehydrogenase activities by ZnO nano-fertilizers, which indicated improved microbial activities in the rhizosphere and the resultant nutrient mobilization for plant uptake. Pandey [44] also reported improved plant growth as a result of nano ZnO application. We can deduce that zinc nanofiber can offer the potential to mitigate the inherent fixation issue associated with alkaline calcareous soil and improve wheat growth, irrespective of application methods.

4.2. Comparative Effects on Zn Uptake and Use Efficiency

Higher Zn concentration was observed with ZnSO4 foliar in grain and leaf (by 14–16% and 2–13%, respectively) than with ZnSO4 seed priming or soil application. However, the ZnONF foliar spray was 15–17% higher in leaf Zn concentration than the seed coating and approximately equal to seed priming (Table 3). These differences, although statistically similar, indicate a more facilitated translocation of Zn from foliar-applied ZnONF to grain compared to ZnSO4. This might be ascribed to the nano size of the applied ZnONF. The combination of ½ZnSO4 + ZnONF foliar showed 11% higher (p ≤ 0.05) grain but 25% less leaf Zn concentration than its counterpart treatments (Table 2). The effectiveness of foliar Zn application alone or in combination with ZnSO4 for grain Zn content was evident from our results compared to other application modes. Elshayb et al. [43] supported our results and reported significantly (p ≤ 0.05) higher Zn uptake with a mixture of ZnONP and ZnSO4. The Zn controlling factors, such as carbonate content and high soil pH (Table 1) or the generally prevalent nutrient exhaustion in cereal-growing soils, could support a higher crop response to the combination of ZnONF and ZnSO4. Furthermore, Zn foliar spray near the heading stage could result in maximal absorption and utilization for translocation to grain for grain development. Grain Zn concentration with ZnSO4 soil addition and seed priming and ZnONF seed coating and priming were comparable (Table 2), suggesting ZnONF is a useful substitute for conventional ZnSO4 fertilizers. Moreover, the combined application of ZnONF foliar spray or seed priming with ½ZnSO4 soil addition surpassed all other modes of application in terms of Zn use in wheat grains and could be adapted for successful wheat grain Zn biofortification. Previous works such as [45] recommended ZnO nanoparticle seed priming for higher growth, photosynthesis, and yield parameters than control. Saleem et al. [46] reported a significant increase in wheat grain Zn content and yield by applying Zn fertilizers. Since Zn transfers from vegetative parts to developing grains, Zn presents transport through the phloem; therefore, in addition to soil factors such as high pH and calcareousness, the availability of water from soil could have affected the Zn content in grain from soil-applied ZnSO4 or seed coated and primed ZnONF, while this problem can have little effect in the case of foliar application since, being accompanied with water, the foliar applied Zn has considerably swift movement in wheat [35].
Higher Zn concentration in roots and stem for ZnSO4 foliar (Table 3) shows ready mobility of Zn from source (leaves) to sink (stem and onward to roots). The root and stem Zn concentration in the case of ZnONF also confirmed that Zn travels from foliar spray towards the root and from seed priming towards the upper parts since root Zn concentration in the case of seed priming of ZnONF is significantly (p ≤ 0.05) lower than the other two application modes. Irrespective of the Zn source, this trend of Zn movement indicates a source–sink relationship, and the travel is always from source (foliar application) to sink (grain and roots) and vice versa for seed priming, soil application, and seed coating. Previous research [47] also revealed that Zn influx into the plant is concentration-dependent, suggesting it is carrier-mediated and metabolism-dependent, and its uptake from the soil into the root and translocation to shoots indicate its movement across root cells’ plasma membranes. This might also be true for seed priming, seed coating, and combined ½ZnSO4 + ZnONF foliar spray, which might have enhanced Zn translocation from roots to shoots [48].
Results for grain Zn content indicated the maximum and significantly higher grain Zn content (by 157%) with combined ½ZnSO4 and ZnONF (foliar spray), followed by sole ZnONF foliar spray (by 92%), in leaf Zn content for combined ½ZnSO4 and ZnONF (seed coating) (73%), in root Zn content (149%) for sole ZnSO4 (foliar spray and in stem Zn content for sole ZnONF (seed priming) (185%) (Table 4). While all these methods variably affected Zn accumulation in different parts of the crop, foliar application of ZnONF in combination with ½ ZnSO4 as soil addition surpassed the rest of the methods for Zn fortification of wheat grain. The maximum and significant increase in total Zn uptake with ½ZnSO4 + ZnONF seed priming (117%) might be due to more biological yield and, therefore, does not stand as a suitable marker for wheat Zn biofortification. However, improved Zn uptake efficiency (ZUE) through ZnONF seed priming (34%) followed by its seed coating (23%) also shows that these treatments are suitable for application to wheat crops. Application of the sole ZnSO4 (2–3%) in either method or its half-dose addition to soil combined with ZnONF (4–6%) in either method did not improve ZUE in wheat crops.
The lowest Zn uptake from ZnSO4 (Table 4) may explain the lowest Zn accumulation in grain. In the case of the combination of ZnONF seed priming and coating along with ½ZnSO4 as soil, a lower concentration of root Zn might have resulted in low Zn uptake from soil and upward translocation and, resultantly, lower grain Zn fortification than the foliar application. Our results were consistent with [45], suggesting ZnO nano-fertilizer with higher Zn uptake and accumulation in various plant parts; however, the effect of ZnO nanofiber and its most suitable application method on Zn concentration and content in various plant parts was never reported. Our results in this regard found that the application of ZnO nanofiber relative to a conventional (ZnSO4) Zn source significantly improved crop Zn nutrition and grain biofortification in wheat crops. The results are in agreement with the findings of [49], while according to [50], the application of Zn-EDTA and ZnSO4.7H2O significantly enhanced the Zn use efficiency of rice over ZnCl2, Zn3(PO4)2 and oxide. However, the Zn use efficiency with Zn EDTA was found to be significantly superior to ZnSO4.7H2O. Further studies on understanding the mechanisms of Zn nanofiber uptake by crops in different soils with different application methods would provide comprehensive information to optimize this potential efficiently (Figure 4).

5. Conclusions

Wheat growth, yield, nutritional, and Zn uptake traits improved as compared to control with Zn nutrition applied. As compared to conventional Zn source (ZnSO4), ZnO nanofiber (ZnONF) was required in significantly lower quantity (0.5 kg ZnONF-Zn ha−1 vs. 5.5 µg kg−1 of soil ZnSO4-Zn ha−1) but improved wheat growth Zn uptake and quality with various application methods. Specifically, ZnONF applied as seed priming and foliar spray produced more yield than its application as seed coating. Furthermore, the combined ZnONF (foliar spray, seed coating, and seed priming) and ½ZnSO4 maximized the Zn nutrient accumulation in different parts of the wheat. In particular, the ½ ZnSO4 + ZnONF through foliar application attained the highest Zn uptake in wheat grain. Future research must focus on the safety, bioavailability, and toxicity of various NFs and NPs utilized for improving crops. Moreover, ZnO nanofiber (ZnONF) application may be tested under abiotic stresses such as drought, salinity, etc., for inducing tolerance in crops and enhancing crop yields under stress conditions.

Author Contributions

W.A., Z.Z. and F.M., Conceptualization and conduct the research, W.A., M.A. (Muhammad Awais) and F.M. Original manuscript draft preparation and review, M.A. (Muhammad Awais), A.K., J.N. and H.K., methodology, M.A. (Masood Ahmad), S.A., I.A. and M.S.K. software, W.A. and F.M., validation, F.M., M.S.K., J.N. and W.A., formal analysis, M.A. (Muhammad Awais), Z.Q., M.S.K., H.K. and W.A., investigation, Z.Z. and W.A., resources, M.A. (Muhammad Awais) and W.A., writing—original draft preparation, F.M., I.A. and Z.Q., writing—review and editing; F.M., W.A., A.K. and M.A. (Muhammad Awais); visualization, Z.Z. and W.A., supervision, Z.Z., W.A., S.A., F.M. and M.S.K., project administration. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Not applicable.

Acknowledgments

All authors would like to acknowledge the facilitation of this research by the Department of Soil and Environmental Sciences, the University of Agriculture, Peshawar, AMK Campus, Mardan, for providing the research and laboratory support. We would also like to appreciate the support provided by the Department of Applied Physics at the Federal Urdu University of Science and Technology (FUUST), Islamabad, Pakistan, during electrospinning for the preparation of Zn nanofibers. We would like to recognize the support received from the Soil and Water Sciences Laboratory at the Indian River Research and Education Center (IRREC), University of Florida, Fort Pierce, Florida, for the completion of this project.

Conflicts of Interest

All authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Figure 1. XRD spectra of ZnO nanoparticles used in the experiment. For convenient comparison, the abscissa and ordinate of XRD patterns are displayed at the same row under the same condition and are scaled to the same range.
Figure 1. XRD spectra of ZnO nanoparticles used in the experiment. For convenient comparison, the abscissa and ordinate of XRD patterns are displayed at the same row under the same condition and are scaled to the same range.
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Figure 2. SEM images of the ZnO (a) nanoparticles at 10 µm, (b) nanoparticles at 1 µm, (c) nanofibers at 10 µm, and (d) nanofibers at 1 µm.
Figure 2. SEM images of the ZnO (a) nanoparticles at 10 µm, (b) nanoparticles at 1 µm, (c) nanofibers at 10 µm, and (d) nanofibers at 1 µm.
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Figure 3. Pictorial view of the experiment at different stages (Source: Authors) (a) (20DAS), (b) (75DAS), (c) (130DAS), and (d) (155DAS) representing the periodic growth stages of wheat in pots.
Figure 3. Pictorial view of the experiment at different stages (Source: Authors) (a) (20DAS), (b) (75DAS), (c) (130DAS), and (d) (155DAS) representing the periodic growth stages of wheat in pots.
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Figure 4. Graphical presentation of increased wheat production and Zn biofortification in wheat.
Figure 4. Graphical presentation of increased wheat production and Zn biofortification in wheat.
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Table
Table 1. Pre-sowing characteristics of the soil used in the experiment.
Table 1. Pre-sowing characteristics of the soil used in the experiment.
ParametersUnitValue
Texture-Silty loam
pH (1:5 H2O)-7.7
EC (1:5)(dS m−1)0.467
OM (%)1.13
Lime (CaCO3)(%)9.4
Total soil N (%)0.22
AB-DTPA Ext. Znmg kg−10.81
AB-DTPA Ext. Pmg kg−15.77
AB-DTPA Ext. Kmg kg−1102.6
Table
Table 2. Plant height, spike length, spikelets per spike, and grains per spike of wheat as affected by different modes and sources of Zn nutrition.
Table 2. Plant height, spike length, spikelets per spike, and grains per spike of wheat as affected by different modes and sources of Zn nutrition.
TreatmentsPlant HeightSpike LengthSpikelets Spike−1Grain Spike−1
Control78 b10.517.7 c39.3 d
ZnSO4(f)86 ab11.318.7 bc47.0 b
ZnSO4(s)92 a11.219.3 abc51.0 ab
ZnSO4(p)88 ab11.719.3 abc44.3 c
ZnONF(f)82 ab11.019.0 abc43.7 c
ZnONF(c)92 a12.020.7 a52.3 a
ZnONF(p)94 a11.820.7 a52.0 a
½ ZnSO4(s) + ZnONF(f)85 ab11.320.7 a49.0 b
½ ZnSO4(s) + ZnONF(c)92 a12.120.0 ab42.0 c
½ ZnSO4(s) + ZnONF(p)92 a11.218.7 bc50.3 ab
LSD (p ≤ 0.05)12ns1.82.6
Suffix f, s, p, and c means application through foliar, soil addition, seed priming, and seed coating, respectively. NF: nanofiber, means with different letters are statistically significant at p ≤ 0.05.
Table
Table 3. Zn concentration in leaf, stem, root, and grain of wheat as influenced by different modes and sources of Zn nutrition.
Table 3. Zn concentration in leaf, stem, root, and grain of wheat as influenced by different modes and sources of Zn nutrition.
TreatmentsZn Concentration (µg g−1)
GrainLeafRootStem
Control20.8 d17.8 d21.5 f11.1 d
ZnSO4(f)29.0 ab21.2 abc53.5 a17.5 b
ZnSO4(s)25.6 bc20.8 abc26.3 ef15.0 c
ZnSO4(p)26.0 bc18.8 cd36.0 bc16.6 bc
ZnONF(f)29.3 ab18.9 cd34.1 cd11.7 d
ZnONF(c)26.1 bc20.0 bcd37.4 bc16.6 bc
ZnONF(p)25.7 bc19.1 cd29.3 de20.4 a
½ ZnSO4(s) + ZnONF(f)32.9 a18.9 cd38.4 bc12.9 d
½ ZnSO4(s) + ZnONF(c)25.7 bc22.4 ab28.0 e16.5 bc
½ ZnSO4(s) + ZnONF(p)31.1 a23.3 a40.8 b20.2 a
LSD (p ≤ 0.05)3.92.75.42.1
Suffix f, s, p, and c means application through foliar, soil addition, seed priming, and seed coating, respectively. NF: nanofiber, means with different letters are statistically significant at p ≤ 0.05.
Table
Table 4. Zn content in different parts of the crop and total Zn uptake as affected by different modes and sources of Zn nutrition.
Table 4. Zn content in different parts of the crop and total Zn uptake as affected by different modes and sources of Zn nutrition.
TreatmentsZn Uptake (µg pot−1)ZUE (%)
GrainLeafRootStemTotal Uptake
Control148 d84 b75 d119 d427 d
ZnSO4(f)242 bcd127 ab188 a220 bc776 ab3 c
ZnSO4(s)233 bcd114 ab76 d211 c633 bcd2 c
ZnSO4(p)211 cd105 ab103 cd224 bc642 bcd2 c
ZnONF(f)215 cd84 b91 cd126 d516 cd7 c
ZnONF(c)232 bcd123 ab132 abcd235 bc722 abc23 b
ZnONF(p)283 abc111 ab113 bcd340 a847 ab34 a
½ ZnSO4(s) + ZnONF(f)380 a129 ab139 abc190 cd838 ab5 c
½ ZnSO4(s) + ZnONF(c)252 bcd145 a87 cd247 bc731 abc4 c
½ ZnSO4(s) + ZnONF(p)334 ab139 a162 ab291 ab926 a6 c
LSD (p ≤ 0.05)1065258752598.0
Suffix f, s, p, and c means application through foliar, soil addition, seed priming, and seed coating, respectively. NF: nanofiber, ½ ZnSO4 soil: half of the recommended ZnSO4 applied to soil. Means with different letters are statistically significant at p ≤ 0.05.
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MDPI and ACS Style

Ahmad, W.; Zou, Z.; Awais, M.; Munsif, F.; Khan, A.; Nepal, J.; Ahmad, M.; Akbar, S.; Ahmad, I.; Khan, M.S.; et al. Seed-Primed and Foliar Oxozinc Nanofiber Application Increased Wheat Production and Zn Biofortification in Calcareous-Alkaline Soil. Agronomy 2023, 13, 400. https://doi.org/10.3390/agronomy13020400

AMA Style

Ahmad W, Zou Z, Awais M, Munsif F, Khan A, Nepal J, Ahmad M, Akbar S, Ahmad I, Khan MS, et al. Seed-Primed and Foliar Oxozinc Nanofiber Application Increased Wheat Production and Zn Biofortification in Calcareous-Alkaline Soil. Agronomy. 2023; 13(2):400. https://doi.org/10.3390/agronomy13020400

Chicago/Turabian Style

Ahmad, Wiqar, Zhiyou Zou, Muhammad Awais, Fazal Munsif, Aziz Khan, Jaya Nepal, Masood Ahmad, Sultan Akbar, Ijaz Ahmad, Muhammad Shahid Khan, and et al. 2023. "Seed-Primed and Foliar Oxozinc Nanofiber Application Increased Wheat Production and Zn Biofortification in Calcareous-Alkaline Soil" Agronomy 13, no. 2: 400. https://doi.org/10.3390/agronomy13020400

APA Style

Ahmad, W., Zou, Z., Awais, M., Munsif, F., Khan, A., Nepal, J., Ahmad, M., Akbar, S., Ahmad, I., Khan, M. S., Qamar, Z., & Khan, H. (2023). Seed-Primed and Foliar Oxozinc Nanofiber Application Increased Wheat Production and Zn Biofortification in Calcareous-Alkaline Soil. Agronomy, 13(2), 400. https://doi.org/10.3390/agronomy13020400

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