1. Introduction
Maize (
Zea mays L.), a high nutrient demanding crop, requires balanced nutrition, particularly the requirement for micronutrients such as zinc is substantial [
1,
2]. The zinc requirement is manifested as high susceptibility of maize to exhibit zinc (Zn) deficiency disorders and, therefore, maize is considered as a Zn deficiency indicator plant [
3]. The Zn requirement for improved plant growth and development is well established [
2]. It plays a vital role in the production of biomass through its influence on diverse physiological and metabolic processes such as chlorophyll formation, fertilization, and germination [
4]. Biochemically, Zn is an essential and integral component of the cytochrome complex and is also required for nucleotide synthesis and membrane integrity [
5]. At the plant physiology scale, Zn is needed as a cofactor or complexing ion for efficient working of all the six groups of enzymes [
6]. Furthermore, Zn is required as a precursor for the synthesis of the auxin phytohormone [
7] and thus has a role in the promotion of vegetative growth in plants. These studies invariably establish the benefits and the requirement of Zn for plants. Therefore, Zn deficiency will lead to cessation or improper functioning of various metabolic and physiological processes in crop plants.
Soils exhibiting low zinc availability are prevalent in tropical and semi-tropical countries affecting approximately 50% of the land across globe. Intensive cultivation of cereal crops in low organic carbon containing light or sandy and high pH calcareous soils has been responsible for the low available Zn contents. The low soil Zn availability leads to the appearance of characteristic zinc deficiency symptoms in crop plants which includes yellowing of leaves, stunted plant growth, and reduced grain yield [
8,
9]. Therefore, the application of water-soluble Zn fertilizer such as zinc sulfate (ZnSO
4) as a basal dose is a common practice to fulfill the Zn requirement of crops [
10]. However, quick Zn fixation/precipitation due to interaction with the calcium (Ca
2+), phosphate (PO
42−) and carbonate (CO
32−) ions in the soil [
11] and leaching (horizontal/vertical soil zones) particularly in sandy soils [
12] from the soil surface after application of conventional Zn fertilizers renders Zn unavailable for uptake by the crop plants [
13]. A possible alternative can be the development and use of slow or controlled release Zn-formulations besides the nano-scale Zn formulations [
6,
14,
15].
Zinc oxide nanoparticles (ZnONPs) are the most common nano-Zn formulations that have been prepared, applied, and evaluated for their plant growth-promoting properties in recent years [
16,
17]. The mode of application of ZnONPs involves soil, foliar, and seed treatments [
18]. Among these application modes, the seed invigoration treatments, including seed priming and coating with ZnONPs are considered relatively eco-safe and economically prudent [
18]. These seed treatments include supplementation of ZnONPs at low concentrations leading to adsorption of ZnONPs on the surface of the seed coat followed by their penetration and movement through seed tissue layers which significantly improves germination and crop biomass [
17]. Therefore, seed priming and coating is a targeted delivery approach to enhance the availability of micronutrients or other elements during the early growth of the plants [
19]. Cultivation practices combining novel seed invigoration technologies and precision farming can improve the seed germination potential, seedling vigor and development, photosynthesis, and reproductive growth to ensure enhanced yield [
20,
21,
22].
Several recent reports identified the positive impact of foliar application of nano-Zn formulations in different cereal and horticultural crops [
16,
17,
18,
19,
22]. These reports indicate a clear trend for the development of nano-based seed invigoration treatments possibly due to bio- and eco-safety aspects attached to handling and foliar application of the nano-Zn particles which could be better addressed through seed invigoration techniques. The present research intervention involves the synthesis of ZnO nanoparticles by the wet chemistry technique. The synthesized ZnONPs were characterized to establish their nano-scale dimensions and properties through diverse spectroscopy and microscopy techniques. The characterized ZnONPs nano-formulations and the conventional zinc sulphate (ZnSO
4) fertilizer were then utilized as seed priming and coating treatments to evaluate the efficiency of the nano-scale Zn fertilizer and its effect on the fodder maize crop grown under field conditions. Further, the work compares the relative impact of ZnSO
4 fertilizer and ZnONPs and their seed application techniques, i.e., as seed priming and coating treatments for the vegetative growth, and yield attributes besides soil chemical and microbiological characteristics in maize (
Zea mays variety J-1006) crop grown under field conditions.
4. Discussion
The synthesized ZnONPs on microscopy and spectroscopy characterizations exhibited peculiar properties indicating the nano-dimensions. The morphological characterization through SEM and TEM elucidated the occurrence of semi-spherical shape and small size (10 to 40 nm) dimensions of the ZnONPs. Similar morphology of the synthesized ZnONPs has also been reported by Urbina et al. [
6]. The SEM-EDS results corroborated the formation of ZnONPs as indicated through the presence of the characteristic X-ray peaks. The UV-Vis spectroscopy analysis of the ZnONPs showed a sharp absorbance peak at 210 nm wavelength. A similar UV absorbance peak at 202 nm has been reported for the sol–gel method-derived ZnONPs [
23]. The single sharp peak is indicative of the nano-scale range and uniform size distribution of the ZnO particles in the aqueous suspension [
6]. The Fourier Transform Infrared Spectroscopy (FT-IR) showed the occurrence of different functional groups derived from the solvent or other chemicals used for the preparation of the NPs to exist on the surface of the synthesized metal oxide nanoparticles. Metal oxides such as ZnO commonly exhibit characteristic IR absorption peaks in the fingerprint region of the FT-IR spectra of the prepared ZnONPs. Chemingui et al. [
30] have observed a wide peak in the range of 3000 to 3500 cm
−1 due to the O-H bond vibration on FTIR analysis of the synthesized ZnONPs. The carbonate anions (O-C-O) symmetric and asymmetric vibrations besides the lattice vibrations have also been reported [
31]. The three spectra contain the absorption peaks between 1100 and 900 cm
−1 which can be ascribed to Zn-OH vibrations [
32] particularly the peaks at 1018.5 and 1020.7 cm
−1 in FTIR spectra of zinc acetate precursor salt and ZnONPs, respectively, indicate the corresponding Zn-O bond vibrations.
Application of ZnONPs through seed priming and coating treatments enhanced the vegetative growth in field-grown fodder maize including improved plant height and number of plants per plot. This improvement may be attributed to the role of Zn in the production of tryptophan—the precursor of indole-3-acetic acid phytohormone [
33]. ZnO nanoparticles can also modulate phytohormone biosynthesis of cytokinins and gibberellins which can lead to an increase in the number of internodes per plant [
33]. Furthermore, enhanced cell elongation can lead to an increase in plant height in the early stages of plant development [
34]. Similarly, Mahdieh et al. [
35] have observed maximum enhancement in plant height in pinto bean (
Phaseolus vulgaris L.) on seed treatment with ZnONPs (0.15%). The photosynthetic pigments (total chlorophyll and carotenoids) were also enhanced on ZnONP seed treatments. Similar improvement in chlorophyll content has also been reported by Faizan et al. [
36]. They have observed maximum chlorophyll content (measured as SPAD value) in tomato plant on ZnONPs treatment (8 mg L
−1).
The applied ZnONPs ensured an increase in yield due to improvement in the yield attributing characters of the treated plants. The yield increase can be associated with the increase in the chlorophyll content leading to improved photosynthetic efficiency that can be specifically correlated with the enhancement in the soluble protein content, starch content, and dry mass [
37].
The macronutrients (NPK) and micronutrient, i.e., Zinc (Zn) contents in shoot and roots of fodder maize were also enhanced by seed priming and coating with ZnONPs. This may be due to improved development of the root system observed as increased root biomass and length, thereby increasing the nutrient uptake. These results are also in accordance with the study of Kolencik et al. [
37] who have reported significantly highest nitrogen content (17611 mg kg
−1) for ZnONPs (2.6 mg L
−1) treatment compared to the control treatment in foxtail millet (
Setaria italica L.). Similarly, foliar application of ZnONPs (30 mg L
−1) in common bean plants increased the P and Zn content of leaves up to 0.34 and 23.9%, respectively, compared to the control treatment [
38]. Adhikari et al. [
39] have also observed the highest content of Zn (38 mg kg
−1) in root and shoot tissues (30 mg kg
−1) of maize treated with 0.5 ppm zinc oxide nanoparticles.
The forage maize fodder quality parameters are critical for its use as fresh feed for the livestock. Improvement in the yield and quality of the fodder maize may be attributed to improved photosynthesis and other metabolic processes in maize [
34]. Likewise, enhancement in the yield and quality of forage sorghum was observed by the application of micronutrients such as iron, zinc, and manganese [
40]. Sharifi et al. [
34] compared the foliar and seed priming treatments of bulk and nanoformulations of zinc and iron micronutrients and reported improved plant quality parameters including the P-content, biomass, crude protein, and soluble carbohydrates.
Priming and coating with different concentrations of ZnONPs significantly influenced the nutrient concentration in soil. This may be ascribed to nano-scale dimensions of the nano-ZnO formulation which improved the solubility and dispersion of insoluble nutrients in soil, leading to decreased soil absorption and fixation, and increased bioavailability [
34]. The soil macronutrient like available nitrogen (N), Phosphorous (P), Potassium (K), and zinc (Zn) micronutrient contents were also increased in the rhizosphere soil of plants treated with ZnONPs. The increased soil available K, P and Zn contents in the rhizosphere soil may be attributed to enhanced root growth, higher production of the organic acids by the plant roots [
41] and occurrence of P-solubilizing microbes [
42] as indicated through greater alkaline and acid phosphatase activities which cumulatively led to decrease in the pH in near vicinity of the growing roots thus facilitating improved soil available K, P and Zn contents. The enhancement in the soil N content in plants treated with ZnONPs must be due to enhanced microbial biomass or activity particularly the non-symbiotic N-fixer populations [
43,
44] which were increased due to seed priming and coating treatments. These microbes could have led to fixation of the atmospheric N. Additionally, the higher dehydrogenase activity in the ZnONPs priming and coating treatments indicate accumulation of organic N in the form of microbial biomass [
44]. Furthermore, as the actinobacterial population was also increased in these treatments, there will also be rise in the soil protease and urease activities [
45] leading to higher N-transformation and availability though the same were not quantified in this report. Raliya and Tarafdar [
46] and Raliya et al. [
47] have also recorded enhancement in the microbial population and soil enzyme activities by the application of ZnONPs leading to mobilization of essential nutrients, i.e., soil N content. Zinc oxide nanoparticles also affect the soil P contents. An increased amount of residual P in the soil was observed after the harvest of sorghum plants in ZnONPs and zinc salt treatments compared to the control treatment [
48]. The application of ZnONPs in the soil led to increased Zn concentrations in soil under maize cultivation [
49].
The soil microbiological characteristics were also influenced by seed coating/priming treatments of maize seeds. The enhancement in the soil enzyme activities particularly the dehydrogenase enzyme activity—the enzyme involved in respiration and food mobilization can be attributed to the role of Zn as a cofactor of the dehydrogenase enzyme [
50]. Similarly, Kumar et al. [
51] have observed significantly higher dehydrogenase activity (18.78 μg TPF formed/g of soil/h) on the application of nano-ZnO (40 mg kg
−1) as compared to the control treatment. Moreover, significantly increased acid and alkaline phosphatase activities have been reported on application of nano-ZnO (10 mg L
−1) in the rhizosphere of the cluster bean plant [
46]. The soil microbial viable counts were also significantly increased in soil sampled from field plots belonging to ZnONPs priming and coating treatments. Kumar et al. [
51] have reported similar results on the application of nano-ZnO. Likewise, Raliya and Tarafdar [
46] have shown enhanced microbial populations on treatment with nano-ZnO (10 mg L
−1) in the rhizospheric soil of cluster bean.
The PCA results of this study showcased a strong association for vegetative, yield attributing, soil nutrient, and microbiological characteristics with both seed coating and priming treatments of Zn-source particularly ZnONPs. These results are in accordance to the study of Popovi´c et al. [
52] who have observed improvement in the seed emergence, plant height, spike length and grain weight plant
−1 traits in wheat which were found to be associated with the seed priming treatment with ZnONPs. Likewise, another PCA analysis on the effect of soil application of urea crystals coated with
Bacillus sp. augmented ZnO particles on wheat depicted that this treatment was the second best after the zinc sulphate coated urea treatment in the PCA score plots. Additionally, in a pot-culture study of Aziz et al. [
53] on perennial ryegrass, the PCA revealed that the soil chemical and microbiological traits besides the dry matter and plant nutrient status were very well correlated and associated with ZnO nanoparticle treatment.