Seed treatment with iron pyrite (FeS₂) nanoparticles increases the production of spinach

Abstract

Certain nano-materials are known to have plant growth promoting effects, which could find applications in agriculture. We drew inspiration from the nano-factories of deep-sea hydrothermal vents; where iron pyrite nanoparticles serve as fertilizer to sustain chemoautotrophic life forms. We synthesized such iron pyrite nanoparticles in a controlled environment and used them as seed treatment agent (Pro-fertilizer). For us, the term ‘pro-fertilizers’ represents those materials that cause enhanced plant growth with minimum interference to the soil ecosystem when used for seed treatment. We conducted multi-location field trials on spinach crops, since it is a globally popular crop, consumed as both fresh (salads) and processed food. The spinach seeds were treated for 14 hours in an aqueous suspension of iron pyrite nanoparticle (FeS₂ + H₂O) and thereafter directly sown in the field setup for the experiment. The control seeds were only treated in water for the same duration and sown directly in the field. After 50 days, the crop yields from iron-pyrite nanoparticle treated seeds and control seeds were evaluated. The plants developed from iron pyrite nanoparticle treated seeds exhibited significantly broader leaf morphology, larger leaf numbers, increased biomass; along with higher concentration of calcium, manganese and zinc in the leaves when compared to the plants developed from control seeds. We further investigated the possible mechanism resulting in the biomass enhancement following seed-treatment. Our results indicate that there is an enhanced breakdown of stored starch in the iron pyrite treated seeds resulting in significantly better growth. This raises the possibility of developing iron pyrite nanoparticles as a commercial seed-treatment agent (pro-fertilizer) for spinach crops.

---

Introduction

The projected global population will be approximately nine billion by 2050. To provide healthy nutrition to this projected population; agricultural production will have to be increased by about 60%.¹ This increased need mandates the development of innovative and sustainable agricultural strategies. A few such strategies are: application of advanced organic fertilizers, reclaiming waste land, efficient water use, utilizing water bodies for food production, utilizing long forgotten grains,¹ effective use of genetically modified crops,² synthesizing food in bioreactors, use of nano-materials in veterinary medicine,³ integrated pest management⁴ and plant nutrient management.^5,6

Agricultural productivity directly depends on an optimal plant nutrient management system. The past few decades of intensive crop production strategies have resulted in excessive use of chemical fertilizers, which in turn resulted in deteriorating soil health and increasing water pollution.⁷ Strategists, planners and thinkers of modern agricultural practices are pondering the question of ‘How to reduce the use of chemical fertilizers without compromising production?’ In other words, how to develop a sustainable strategy for fertilizer usage.

One approach could be to reduce the size of fertilizers to nano-dimensions so that high surface area to volume ratio can be achieved; where altered surface properties will reduce the dose requirements. This makes nano-fertilizers more advantageous over conventional fertilizers.^5,6 In addition, this will reduce the cost of fertilizers and will significantly reduce soil–water pollution. Effectiveness of nano-strategy in developing slow release of fertilizer is reported in case of potash fertilizers⁸ and in urea-modified hydroxyapatite nanoparticles for sustained release of nitrogen in the soil.⁹ Carbon nano-structures are also used to increase plant growth.^10,11 Most of these nanoparticles that are being tested for their plant growth promoting effects; are of anthropogenic origin (do not exist in nature) and get directly applied in the growth substrate (soil or water). There is no study till date, which attempted to exploit the potency of any kind of nanoparticle as a seed-treatment agent. Although a wide variety of physical and chemical approaches for seed treatment are documented in the literature; however, the nanomaterial as a seed treatment agent is a comparatively novel approach (ESI: Section S1: Table S1.† Detailed list of different seed treatment agents). If such an approach is successful in increasing the yield, then we can reduce the consumption of fertilizers and reap rewards mentioned earlier. We speculate that during the onset of germination, when the seeds are experiencing an extremely fertile metabolic phase, certain nanoparticles may significantly influence the physiology of plant growth; which essentially becomes the premise of this work. In this work, we develop and demonstrate a seed treatment strategy for spinach crop using iron pyrite nanoparticles, which resulted in significantly higher yield.

The most obvious question is ‘why iron pyrite nanoparticles were chosen for our study?’ We drew inspiration from the hydrothermal vents of nature. In late 1970s, several deep-sea expeditions resulted in the discovery of hydrothermal vents and the amazing life forms (giant tubeworms, shrimp, clams and limpets) surrounding these vents. The ecosystem of hydrothermal vent is devoid of sunlight, has high pressure and temperature, and also is rich in iron pyrite nanoparticles and sulfides. These nanoparticles are naturally synthesized in abundance in the nano-factories of the hydrothermal vents. One of the major question arise out of this discovery was ‘how in such an extreme environment, devoid of light, the life form thrives?’ On further probing, it was discovered that the giant tubeworms, which accounts mostly for the very high population in such vents, symbiotically harbors wide range of chemo-autotrophic microbes in their body. The symbiotic microbes are reported to house proteins that are involved in energy coupling by oxidation of the available metal sulfides; primarily FeS₂.¹² Thus FeS₂ nanoparticle synthesized in the hydrothermal vents act as an energy source for chemoautotrophic life forms.^13–15 It is through this oxidation of sulfides and energy coupling, the higher symbiotic organism i.e. tubeworms obtain its energy for survival. Iron pyrite is also known to be linked with other chemoautotrophic organisms¹⁶ and has great evolutionary significance.¹⁷ Researchers also showed interactions between microbes such as Acidothiobaccilus feroxidans and pyrite surface.¹⁸ Thus FeS₂ nanoparticle function as a fertilizer for sustaining life forms in deep ocean floor and in oxygen deficient environments. These findings inspired us to look for any relationship between the FeS₂ and higher autotrophic life forms.

In order to address this problem, we needed pure iron pyrite nanoparticles. Iron pyrite is abundantly present in the nature, but it is contaminated with arsenic¹⁹ and other heavy metals. Therefore, we developed a simple strategy to synthesize pure FeS₂ nanoparticles for our studies. Keeping in mind that in future, we need to reduce the consumption of commercial fertilizers without compromising on the productivity; we decided to use FeS₂ nano-particles as a seed treatment agent. In one of our recently concluded studies, we have shown that in acute and sterile laboratory conditions; when Cicer arietinum seeds are treated with iron pyrite nanoparticles, and grown for 7 days in pure, sterile water; significantly healthy plants with increased dry-weight were observed.²⁰ Though our preliminary results were promising; but, from the perspective of a farmer, the major question is: ‘will this novel approach of seed treatment with iron pyrite nano-particles will be effective in the field trials?’ Moreover, ‘will this nano-particle based strategy of crop production is economically effective?’

To address these questions, we evaluated the agricultural production of spinach crop (Spinacia oleracea) after seed treatment with FeS₂ nanoparticles. Spinach is popular and global vegetable, which is rich in iron,²¹ calcium²² and many vitamins, mainly vitamin A.²³ Further, spinach leaf extracts has demonstrated anti-proliferative, anti-aging, anti-inflammatory, and anti-oxidant properties in different experimental models.²⁴ We conducted multiple location field trials on this short duration leafy vegetable crop; where harvesting happens after 60 days of sowing. The FeS₂ nanoparticle treated spinach seeds are sown in the fields. After 50 days, we observed that the FeS₂ treated seeds resulted in significantly higher: number of leaves per plant, fresh and dry weight per plant, leaf and leaf area index per plant and higher concentration of calcium, manganese and zinc in the leaves; when compared to control seeds. Further, we proposed a possible mechanism for the observed effects of FeS₂ nanoparticles on the seed physiology. Overall schematic of the study is shown in Fig. 1.

Fig. 1 The outline of the study involving synthesis and characterization of nanoparticles, and using them as seed treatment agent, followed by monitoring of the crop yield.

Results and discussion

Iron pyrite nanoparticle synthesis and characterization

We devised a comparatively low temperature synthesis of iron pyrite nanoparticle. The particle size was controlled using tri-sodium citrate (TSC) as a capping agent. The proposed synthesis strategy is a slight modification of our previous work.²⁰ The synthesized material was then characterized using X-ray diffraction (XRD) technique and diffraction pattern is shown in Fig. 2a. The 2θ peaks at 28.4, 32.8, 36.9, 40.6, 47.3, 56.03 and 58.7 can be indexed to planes (111), (200), (210), (211), (220), (311) and (222), which is in consistency with the iron pyrite structure (JCPDS no. 42-1340); conforming the formation of iron pyrite nanoparticles. The SEM images (Fig. 2b and c) showed a pitcher like morphology of the particles. The size of the particles ranges from 600–700 nm. The synthesized iron pyrite nanoparticles are of uniform shape, size and morphology.

Fig. 2 Characterization of synthesized iron pyrite nanoparticle. (a) XRD (b and c) SEM images of the particles showing pitcher like morphology.

Fig. 3 X-ray photoelectron spectra of iron sulphide (FeS₂) nanoparticles. (a) The core Fe2p spectra of the freshly prepared dry FeS₂ sample. (b) The Fe2p spectra after exposure to water. (c) The core S2p spectra of the freshly prepared dry FeS₂ sample. (d) The S2p spectra after exposure to water.

XPS analysis of the FeS₂ nanoparticles

XPS analysis was performed to further verify the composition of the FeS₂ nanoparticles in their native state as well as upon exposure to water (Fig. 3). The XPS spectra of both the native samples and the water exposed samples show, two major peaks at 707.5 eV and 720.1 eV due to the Fe2p_3/2 and Fe2p_1/2 spin–orbit coupling. The peak at 709.1 eV may be due the defect on the surface of FeS₂. De-convoluted XPS of FeS₂ sample after exposure to oxygen deficient water indicate the formation of FeO, Fe₂O₃, FeOOH and FeSO₄ at binding energy of 710.1 eV, 711.0 eV and 712.0 eV. Further a significantly enhanced signal of Fe₂(SO₄)₃, FeSO₄ and SO₂ was observed in S2p spectra of water exposed sample. Upon exposure to water, noticeable structural changes were observed in the FeS₂ nanoparticles. In the overall subsequent sections, we have highlighted some of the implications of these reported surface defects on the iron pyrite surface.

Effect of FeS₂ on the emergence of spinach seed

We initially investigated the percentage emergence of spinach seeds. In order to compare and quantify the effects of different nanoparticles on the emergence of seeds; we selected three different nanoparticles, viz., iron pyrite (FeS₂), cerium oxide and graphene oxide. Along with this, we quantified the effects of different salts namely Fe²⁺ salt, Cerium salt and charcoal. We observed significantly higher emergence, when the seeds were treated with FeS₂ (ESI section S2†). This experiment demonstrated that significantly higher seed emergence is exhibited solely by FeS₂ nanoparticles when compared to other nanoparticles and their corresponding salts. This led us to conduct the field trials using FeS₂ treated spinach seeds.

Plant growth experiments with FeS₂ nanoparticles

Equal amount of the spinach seeds were randomly divided into 2 groups: (i) control, and (ii) test. The control group seeds were soaked in sterile, double distilled water for 14 hours before sowing; whereas, the test group seeds were soaked in a suspension of double distilled water + FeS₂ particles for 14 hours before sowing them in the field. At the time of harvest, various parameters pertaining to the yield and biomass were calculated. It was observed that control plants had average number of leaves about 13 ± 1; whereas, the test plants had average number of leaves at 19 ± 1 (Fig. 4a). Specific leaf area was calculated and the data was found corroborating with the data obtained upon measuring the thickness of the leaves (Fig. 4b). Also, Fig. 4c is the arial view of the field showing that FeS₂ treated seeds resulted in significantly more foliage.

Fig. 4 Plant growth parameters: control versus test (FeS₂). (a) Number of leaves/Plant: control: 13 ± 1.0; test: 19 ± 1.0. (b) Specific leaf area signifies leaf thickness and was found similar for both test and control samples. (c) Field photograph taken at day 50 (just before harvesting the crop) depicting that the test group plants have comparatively more foliage as compared to the control plants.

Total leaf area was calculated (Fig. 5a) and found to be significantly higher for the test samples. Next, we calculated the ‘Leaf Area Index (LAI)’, which is a measure of the total photosynthetic area available to the plant. Test samples demonstrated significantly higher ‘LAI’ with values of 1.53 ± 0.07, when compared to the control value of 0.94 ± 0.02 (Fig. 5b). Representative pictures of the leaves obtained from the test and the control plants are shown in Fig. 5c. High leaf area index values also hints at high biomass content of the test samples in comparison to the control.

Fig. 5 Plant growth parameters: control versus test (FeS₂). (a) Leaf area/Plant, showing significant increase in leaf area/test plants (52.4 ± 0.3) as compared to control (25.6 ± 0.2). (b) Leaf area index signifying total photosynthetic area available to plant and high values for test samples (1.5 ± 0.07) can be correlated with high biomass content of pro-fertilized spinach plants in comparison with (0.9 ± 0.02). (c) Comparative photograph of leaves showing larger leaf area in test plants as compared to control plants.

Next we evaluated the fresh and dry weight of control and test plants. Test samples showed tremendous increase in both fresh and dry weight, which is indicative of significant increase in biomass of the test sample upon FeS₂ seed treatment (Fig. 6a and b).

Fig. 6 Plant growth parameters: control versus test (FeS₂). (a) Fresh weight comparative graph showing significant increase in biomass in test samples (16.7 ± 0.4) as compared to control (8.4 ± 0.4). (b) Dry weight comparative graph corroborating with high biomass content in test plants. Control: 0.3 ± 0.1; test: 0.7 ± 0.1. (c) Calcium concentration in parts per million (ppm). (d) Iron, manganese and zinc concentration in ppm.

Elemental analysis

The results obtained from spectroscopy showed significant increase in calcium, manganese and zinc in the test samples. However, no significant difference in iron concentration was found between two samples; with mean values 0.1429 ppm with standard error (SE) of 0.0004 for the test, whereas, 0.1406 ppm, SE 0.0017 for the control. Also, values in ppm for calcium, manganese and zinc were 7.621 ± 0.021, 0.6146 ± 0.0008 and 1.151 ± 0.003 respectively for test samples and 5.581 ± 0.017, 0.4862 ± 0.0049 and 0.7286 ± 0.0017 respectively for control samples (Fig. 6c and d). It should be noted that these values for test samples are significantly higher when compared with control samples. While calcium is involved in structural roles as well as cell signaling,²⁵ manganese finds its major role in photosynthesis.²⁶ Also, inadequate zinc is known to reduce crop yields and is essential for plant growth.²⁷ This significant increase in the concentrations of these important nutrients warrants for further investigations in future. Iron concentration does not show any significant change, which is in accordance with our previous study.²⁰ The possible reason why we do not see any significant change in iron concentration is that iron is solely acting as a metal factor in the surface chemical reaction of iron pyrite + water + seed. The mechanism is discussed in the subsequent section.

Dissecting the possible mechanism for the observed seed treatment effects

We attempted to dissect the possible molecular mechanism observed from seed treatment effects through a series of innovative, yet simple experiments. Seeds contain significant amount of stored starch, which is the prime mover for emergence and germination. During germination, seeds derive necessary energy for initial growth by breaking down the stored starch molecules into reducing sugars. This breakdown is enzymatically driven by the family of alpha-amylase enzymes. Thus there is an increase in alpha-amylase activity during germination. The alpha-amylase activity is regulated by Giberellin, a key plant hormone involve in germination and growth of plants.²⁸ This is the most accepted paradigm of seed germination. Fig. 7 highlights the key processes of seed germination. We were curious on the role played by FeS₂ particles in this accepted paradigm of seed germination.

Fig. 7 The major molecular players involved in the germination of the seeds.

We conducted six sets of experiments, with each replicated six times (n = 6) to confirm each of our findings. In the first five cases, the control experiment was seeds treated with double distilled water. For the comparative experiments, the seeds were treated with FeS₂, water, and specific chemicals in certain cases to modulate certain starch breakdown pathways. The experimental layout is given in Fig. 8.

Fig. 8 The overall experimental layout to dissect the mechanism of action of FeS₂ on the germination of seed.

In the first experiment, we collected the leachate after 14 hours of seeds treatment from both control and test group. It was analyzed for the presence of reducing sugar. It was found that the test group leachate was 40% more rich in reducing sugars when compared with the control (Fig. 9a); allowing us to postulate that higher amylase like activity is promoted by FeS₂ treatment of seeds. Next, we tested for amylase activity in control and treated seeds for durations of 14 hours after treatment and fully germinated seeds at day 7. The amylase activity was found to be 10% and 30% more in both cases respectively (Fig. 9b and c). The generally accepted fact is that vigorous amylase activity occurs after 72–96 hours of water imbibition during germination, and hence, the higher amylase activity is observed in the later case (Fig. 9c). To quantify the influence of FeS₂ on amylase activity, we conducted another set of experiments; where the intrinsic activator of amylase in seeds, viz., Gibberellin was blocked using abscisic acid (ABA). After the treatment with ABA, the amylase activity in the germinated seeds was significantly reduced as per the accepted theory. However, we observed that fully germinated seeds treated with ABA and FeS₂ demonstrated slight increase (∼5%) in the amylase activity at day 7 (Fig. 9d). Continuing our investigation, we conducted another set of experiments, where the intrinsic amylase activity was blocked using amylase inhibitor. Interestingly, we found that the fully germinated seeds treated with amylase inhibitor and FeS₂ exhibited ∼20% more amylase activity when compared with fully germinated seeds treated with just amylase inhibitor (Fig. 9e). These results provoked our curiosity, and we conducted a simple starch breakdown assay in a controlled setting to investigate the capability of FeS₂ alone in breaking down starch molecules in the presence of water. In this experiment, the control was starch plus double distilled water; whereas, the test setup contained starch with double distilled water along with FeS₂. Both setups were kept aside for 14 hours at room temperature; after which the percentage of reducing sugars were estimated in both cases. To our surprise, it was found that the test case demonstrated ∼40% more reducing sugar content than the control (Fig. 9f).

Fig. 9 Summary of the experiments to understand the effect of FeS₂ on starch metabolism. All the experiments were repeated 6 times (n = 6) and the data was pooled. The results are reported as mean ± standard error (SE). (a) Total amount of leachate reducing sugars in iron pyrite treated seeds show significant increase (136.23% ± 11.45) as compared to control seeds' leachate sugars. Values for control have been taken as 100% and the amount of absorbance for test is shown in comparison to the same. (b) Total amount of amylase activity in iron pyrite treated seeds after 14 hours of seed treatment show slight increase (108.85 ± 4.48) as compared to control seeds' amylase activity. Values for control have been taken as 100% and the amount of absorbance for test is shown in comparison to the same. (c) Total amount of amylase activity in iron pyrite treated seeds after seeding growth (7 days) show significant increase (132.37% ± 11.57) as compared to control seeds' amylase activity. Values for control have been taken as 100% and the amount of absorbance for test is shown in comparison to the same. (d) Effect of abscisic acid (ABA), a seed dromany inducer and gibberellin blocker was examined on total amylase activity. A significant decrease in total absorbance was observed (79.86% ± 13.03) as compared to control. Also incubation with iron pyrite could not bring about a significant positive change (83.33% ± 12.67). Values for control have been taken as 100% and the amount of absorbance for test is shown in comparison to the same. (e) 5% inhibition in pure amylase activity was observed and thus amylase inhibitor activity was conformed. A marked increase (115.25% ± 7.33) in the amylase activity was also observed in amylase inhibitor + iron pyrite incubated samples. Values for control have been taken as 100% and the amount of absorbance for test is shown in comparison to the same. (f) Starch breakdown in presence of iron pyrite nanoparticles was seen as there was significant increase (130.62% ± 9.26) in the absorbance for reducing sugars. Values for control have been taken as 100% and the amount of absorbance for test is shown in comparison to the same.

The hydrolysis of starch to reducing sugars by FeS₂ in the presence of water, and the continuing starch breakdown to reducing sugar even in the presence of amylase inhibitor suggest that FeS₂ alone can breakdown starch in the presence of water. Thus one can say that FeS₂ could mimic the enzymatic activity of amylase enzyme. So the next pertinent question is ‘How starch could be hydrolyzed by FeS₂ nanoparticles?’ The most plausible reason lies in the intricate surface chemistry of iron pyrite molecule. Iron pyrite surface has ‘iron defect’ sites.^29–31 Further our XPS results highlighted the presence of such surface defects on the iron pyrite nanoparticles (Fig. 3). A surface mediated reaction on these defect sites between pyrite and water (either in the absence or presence of oxygen) leads to the production of significant amount of hydrogen peroxide (H₂O₂).^29–31 The amount of H₂O₂ liberated in such reactions is a direct function of defect site density and available surface area of the particles.^29–31 So unlike the ‘classical Fenton reagent’, which is a mixture of hydrogen peroxide and ferrous salts, there is an in situ production of H₂O₂ on the surface of iron pyrite in the presence of water.^29–31 This attribute of FeS₂ has led scientists to coin the term the ‘pyrite-only Fenton-like’ (PF) reagent.³² Both Fenton and PF reagent are effective oxidants by virtue of their ability to generate highly reactive hydroxyl radicals. PF has been shown to oxidize wide range of organic compounds including lactate,³² carbon tetrachloride,³³ chlorinated ethylenes,^34–37 aromatic nitro compounds³⁸ and copper phthalocyanine.³⁹ Earlier it has been demonstrated that starch could be hydrolyzed by Fenton reagent.⁴⁰ Here we are demonstrating that PF also has the ability to hydrolyze starch. So we could consider iron pyrite nanoparticle system (FeS₂ + H₂O + starch) as the ‘Artificial Enzyme System’ mimicking amylase activity in hydrolyzing starch. Thus when seeds are treated with FeS₂, they have more amylase activity as compared to the control seeds. This enhanced amylase activity and pronounced breakdown of stored starch in the seeds act as a strong growth booster in future development of the plant, as we observe in the field trial.

Next we asked ourselves another question. Is the enhanced biomass of adult plant exclusively due to enhanced amylase activity of the germinated seeds or during that germination phase some other growth promoting pathway might have got triggered? Here we speculate that H₂O₂ generated by FeS₂ + H₂O + seed could act as a chemical messenger. Earlier research has reported that H₂O₂ is involved as a secondary messenger in stimulating brassinosteroids mediated CO₂ assimilation, redox signaling and carbohydrate metabolism.^41,42 Since, FeS₂ can generate hydrogen peroxide in aqueous environment; the brassinosteroid mediated pathway of CO₂ assimilation and carbohydrate metabolism can be enhanced by increased supply of hydrogen peroxide by pyrite. The overall proposed mechanism is outlined in Fig. 10. Thus, the increase in biomass and plant size upon seed treatment with FeS₂, can be attributed to the modulation and increased activity of the aforementioned pathway by H₂O₂ generated by FeS₂.

Fig. 10 Proposed outline of the mechanism of action of FeS₂ on spinach seed in enhancing germination and plant growth.

Materials and methods

Iron pyrite nanoparticle synthesis and characterization (XRD, SEM, XPS)

Here in this report, we present a low temperature synthesis of FeS₂ nanoparticles using tri-sodium citrate as capping agent. The FeS₂ particles were synthesized by reacting FeCl₃ in an acidic buffer of pH 5.6, along with sodium polysulfide (Na₂S_x) under an inert atmosphere. The basis of this reaction was FeS₂ can be synthesized using polysulfide.⁴³ Sodium polysulfide stock was synthesized as described in earlier work. Equal volume (100 ml) of Sodium acetate–acetic acid buffer (pH 5.6) and 0.04 M FeCl₃ were mixed. Argon was purged for 20 minutes to remove any dissolved oxygen in water, and to prevent any oxidation and thereby maintaining an inert environment. To this solution, 100 ml of capping agent i.e. tri-sodium citrate (0.2 M) was added and argon purging was done continuously. 15 ml of sodium polysulfide was added drop-wise to this solution and a black coloration was observed indicative of FeS formation. Following this continuous stirring and heating in an oil bath at 90–100 °C was done for another 4 hours, until black solution turns to grayish in color (Fig. 11). The grayish solution obtained was centrifuged and the precipitate was washed as described in earlier work.²⁰ Powder X-ray diffraction measurements were carried out with a Bruker D8 Advance and a Rigaku miniflex-(II) X-ray diffractometer using monochromatized Cu Kα radiation (λ = 1.54056 Å) at a temperature of 298 K. Scanning electron microscopic (SEM) images were obtained using the SUPRA 40VP field emission scanning electron microscope Carl Zeiss NTS GmbH, Oberkochen (Germany). XPS characterization was performed in the PHI Quantera II Scanning XPS microprobe by using a 100 μm X-ray beam at 100 W and at a base pressure of 5 × 10 ⁻⁸  Torr power raster scanning over a 1400 × 100 μm area of the sample.

Fig. 11 Representative scheme of iron pyrite nanoparticle synthesis. TSC = Tri-sodium citrate; Na₂S_x = sodium polysulfide.

Preparation of the plots for conducting field trials

The plots were randomly chosen in the institute nursery. Each plot, in which trials were conducted, had a dimension of 5 feet × 6 feet. Manual tilling was done and the plots were leveled for proper water distribution. The plots thus prepared were pre-irrigated and left for 3 days for the soil (pH 6.5) to get moistened uniformly. Manual weeding was performed. No insecticide or pesticides were used in these crops. No organic manure or chemical fertilizers were used during the trials.

Plant growth experiments

Spinach was chosen for plant growth experiments due to its economic importance and high nutritive value. Also spinach has been reported to have high iron concentration. Commercially available spinach seeds were chosen to ensure the variability in the seeds. The field trials were carried out at three different locations. On each plot of control and test, 1.5 gram healthy spinach seeds were sown. After conducting three different trials (N = 3) on randomly selected plots, we observed that both in control and test, around 150 seeds germinated to full grown plants (n = 150). The data obtained from three different field trials were pooled to draw the final inferences.

Seed treatment

The synthesized nanoparticles were used for the seed treatment or pro-fertilization of the spinach seeds. This is a novel approach to study the effectiveness of nanoparticle on plant growth. Three grams of seeds were taken for each trial and three field trials were performed. The seeds were initially treated with 10% sodium hypochlorite solution for 10 minutes to ensure surface sterilization¹⁰ and washed 5 times with de-ionized water to remove any traces of hypochlorite. The seeds were then divided into two equal groups viz. control and test. Control seeds were kept overnight in de-ionized water, while the test seeds were kept in an aqueous suspension of synthesized FeS₂ (80 μg ml⁻¹ of water) in 90 mm petri dishes. This dose was optimized in our previous experiments.²⁰ The seeds were pretreated for 14 hours and directly sown into the fields.

Evaluating the plant growth parameters

The following three growth parameters were evaluated: biomass (fresh weight and dry weight), number of leaves per plant, leaf area per plant, leaf area index and specific leaf area.

1. Biomass and number of leaves per plant: the seeds were allowed to germinate and grow in the fields for 50 days and then harvested manually. Total produce was cleared of any mud attached in the roots by washing in running water and weighed to obtain the fresh weight of the samples. Number of leaves in each plant was counted manually and the samples were further subjected to dry heat in an oven at 75 °C for 48 hours. The dried samples were again weighed to get the total dry weight of the produce. This whole procedure was carried out for all the three plots.

2. Leaf area per plant: ten random fresh leaves were taken from each group i.e. control and test, thickness of each leaf in the middle portion was measured with a micrometer, and no significant difference was found. This data was further corroborated with specific leaf area measurement. One square inch area from 10 random leaves was carefully marked and cut with a scalpel. This area was weighed and total plant area was measured by multiplying the fresh weight of one square inch leaf to the total fresh weight of the produce. The leaf area per plant is calculated by dividing the total leaf area by the total number of plants. Photosynthesis is a function of total leaf area and signifies the amount of biomass produced.

3. Leaf area index: leaf area index signifies the total photosynthetic area available to the plant and is calculated as the ratio of total leaf area to the total field area. The formula for calculating ‘Leaf area index’ is [Leaf area index = total leaf area/total field area]. Leaf area index was calculated for all three trials and mean was calculated.

4. Specific leaf area: specific leaf area is calculated as the ratio of leaf area to dry mass. It signifies the thickness of the leaves. The formula for calculating ‘Specific leaf area’ is [Specific leaf area = Leaf area/Dry mass]. The data obtained was found in corroboration with leaf thickness measurements with the help of micrometer.

Elemental analysis

Elemental analysis of dried leaf samples was done using inductively coupled plasma spectroscopy (iCAP 6300 ICP Spectrometer, Thermo). Leaf samples were dried in dry heat oven at 75 °C for 48 hours, these dried samples were subjected to nitric acid digestion for 2 hours, filtered and the filtrate was used for spectroscopy.

Biochemical assays performed to dissect the mechanism of action of FeS₂ nanoparticles on seed

1. Leachate sugars: 1 ml of water in which the seeds were soaked overnight was taken in a test tube and 1 ml of DNSA (di-nitro salicylic acid) was added along with 1 ml of fresh distilled water. The tubes were mixed well and incubated at 90 °C for 10 minutes in a water bath. The solution was allowed to cool down and absorbance was measured at 540 nm after 3× dilution.

2. Crude seed extract: crude seed extract was prepared by grinding the germinated seedlings using a pestle mortar, in 10 ml distilled water along with small amount of silicate. The extract thus obtained was filtered and volume was made upto 50 ml.

3. Amylase activity test: amylase test was performed as reported in previous literature with slight modification.⁴⁴ 1 ml Crude amylase extract was mixed with 1 ml citrate buffer (pH 5.6) and incubated at 40 °C in a water bath for 10 minutes. To this solution, 2 ml of 1% starch solution was added and again kept for incubation at 40 °C for 10 minutes. Further to stop the amylase activity, 4 ml of 0.4 M NaOH was added. 1 ml of this final solution was used for DNSA assay for reducing sugars.

4. ABA (Gibberellin inhibitor): abscisic acid (ABA) is a dormancy inducer and an inhibitor of gibberellin. We used 50 μM of ABA independently and along with FeS₂ during the time of seed treatment. In the control, no ABA was used. ABA was obtained from commercial vendor.

5. Amylase inhibitor extraction from Ragi seeds: amylase inhibitor extraction was done as described by Kumar et al. from ragi seeds.⁴⁴ 80 gram of seeds were grounded in a pestle mortar to fine powder. 240 ml of 0.15 M NaCl was added and stirred for 3 hours at room temperature. The slurry obtained was filtered under vacuum. 52 grams of ammonium sulfate was added to 150 ml of filtrate and was mixed thoroughly. The solution was allowed to stand overnight at 4 °C. This was further centrifuged at 24 000g for 20 minutes and the precipitate was dissolved in 50 ml distilled water. The solution was properly dialyzed against distilled water. Dialyzed solution was centrifuged for 20 minutes at 24 000g. Further 40 ml of clear supernatant was heated at 70 °C for 30 minutes to deactivate amylase and to remove heat labile proteins. The solution was again centrifuged at 24 000g for 20 minutes. The supernatant was carefully taken and stored at 4 °C for further use.

6. Effect of iron pyrite on amylase inhibitor: 2 ml of extracted amylase inhibitor was incubated with 100 μl of iron pyrite suspension (10 mg ml⁻¹) for 3 hours. This was then centrifuged at 5000 rpm for 20 minutes to get a clear supernatant of amylase inhibitor. Similar procedure was done for control with 100 μl distilled water. 1 ml of supernatant was carefully taken in a test tube. 1 ml amylase (1 mg ml⁻¹) and 1 ml citrate buffer (pH 5.6) were added. This mixture was incubated in a water bath at 40 °C for 30 minutes. 2 ml of 1% starch solution was added and incubated at same temperature for 10 minutes. This solution was used to carry out DNSA assay for reducing sugars.

7. Iron pyrite and starch incubation: 2 ml of 1% starch solution was mixed with 1 ml citrate buffer (pH 5.6). 100 μl of iron pyrite suspension was added to the test solution and volume of control solution was made up with 100 μl distilled water (1 mg FeS₂ nanoparticle). This was kept overnight (12 hours) in the dark. After incubation the solution was centrifuged at 5000 rpm for 30 minutes and 2 ml of the supernatant was carefully taken for DNSA assay.

8. DNSA reagent: 1 gram of di-nitro salicylic acid was added to 50 ml water. To this solution 30 grams of sodium–potassium tartrate was added in small amounts. Milky yellow color is observed, which turns to transparent yellow upon addition of 20 ml of 2 M NaOH. Final volume is made to 100 ml and the reagent is kept at 4 °C protected from light. We prepared fresh solution for every individual assay.

Conclusion

We conclude that FeS₂ nanoparticle could function as pro-fertilizer. Simple seed treatment with iron pyrite (FeS₂) nanoparticles increase the production of spinach crop possibly by two different routes. First in the presence of water, it results in in situ generation of hydrogen peroxide, thereby breaking down the starch more rapidly. Thus acting as an artificial enzyme mimicking the amylase activity. Second, it acts as a chemical messenger by activating the brassinosteroid pathway, thereby augmenting CO₂ fixation and carbohydrate metabolism. Such innovative seed treatment strategy promises huge prospects in overcoming soil and water pollution and maintaining the soil ecosystem. Such conservative and judicious use of plant growth promoting nanoparticles as seed treatment agent could emerge as a novel strategy in the domain of sustainable agriculture.

Acknowledgements

We sincerely thank Prof. Tarun Gupta of Department of Civil Engineering, Indian Institute of Technology Kanpur for providing ICP Spectrometer facility. Mr Rajeev Garg, ‘Superintendent Engineer’ and Head of Institute Works Department (IWD) and Institute Nursery for providing all the logistical supports, labor support for helping in conducting the field trials. Further we appreciate all the help from IIT Kanpur administration for encouraging the field trials. This work is part of GS's Doctoral research work. GS's thanks MHRD, IIT K, GOI for his Doctoral research fellowship.

References

1. M. Hogenboom, Creating new life – and other ways to feed the world, BBC News, 22 July 2013.
2. S.-E. Jacobsen, M. Sørensen, S. Pedersen and J. Weiner, Agron. Sustainable Dev., 2013, 33, 651–662.
3. V. P. Chakravarthi and N. Balaji, Vet. World, 2010, 3, 477–480.
4. A. Pérez-de-Luque and D. Rubiales, Pest Manage. Sci., 2009, 65, 540–545.
5. M. C. DeRosa, C. Monreal, M. Schnitzer, R. Walsh and Y. Sultan, Nat. Nano, 2010, 5, 91.
6. R. K. Sastry, H. B. Rashmi, N. H. Rao and S. M. Ilyas, Technol. Forecast. Soc. Change, 2010, 77, 639–648.
7. X. T. Ju, C. L. Kou, P. Christie, Z. X. Dou and F. S. Zhang, Environ. Pollut., 2007, 145, 497–506.
8. G. K. Ch, V. Subbarao and D. Sirisha, International Journal of Applied Science and Engineering, 2013, 11, 25–30.
9. N. Kottegoda, I. Munaweera, N. Madusanka and V. Karunaratne, Curr. Sci., 2011, 101, 73–78.
10. S. Tripathi, S. K. Sonkar and S. Sarkar, Nanoscale, 2011, 3, 1176–1181.
11. S. K. Sonkar, M. Roy, D. G. Babar and S. Sarkar, Nanoscale, 2012, 4, 7670–7675.
12. C. R. Fisher and P. Girguis, Science, 2007, 315, 198–199.
13. J. B. Corliss, J. Dymond, L. I. Gordon, J. M. Edmond, R. P. von Herzen, R. D. Ballard, K. Green, D. Williams, A. Bainbridge, K. Crane and T. H. van Andel, Science, 1979, 203, 1073–1083.
14. M. Yucel, A. Gartman, C. S. Chan and G. W. Luther, Nat. Geosci., 2011, 4, 367–371.
15. R. A. Zierenberg, M. W. W. Adams and A. J. Arp, Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 12961–12962.
16. G. Wachtershauser, Philos. Trans. R. Soc. London, Ser. B, 2006, 361, 1787–1806 ; discussion 1806–1788.
17. G. Wächtershäuser, Syst. Appl. Microbiol., 1988, 10, 207–210.
18. H. Tributsch, S. Fiechter, D. Jokisch, J. Rojas-Chapana and K. Ellmer, Origins Life Evol. Biospheres, 2003, 33, 129–162.
19. J. Matschullat, Sci. Total Environ., 2000, 249, 297–312.
20. G. Srivastava, A. Das, T. S. Kusurkar, M. Roy, S. Airan, R. K. Sharma, S. K. Singh, S. Sarkar and M. Das, Mater. Express, 2014, 4, 23–31.
21. C. H. Schwietzer, Med. Klin, 1960, 55, 1271–1275.
22. L. McLaughlin, J. Biol. Chem., 1927, 74, 455–462.
23. G. Tang, J. Qin, G. G. Dolnikowski, R. M. Russell and M. A. Grusak, Am. J. Clin. Nutr., 2005, 82, 821–828.
24. L. Lomnitski, M. Bergman, A. Nyska, V. Ben-Shaul and S. Grossman, Nutr. Cancer, 2003, 46, 222–231.
25. P. J. White and M. R. Broadley, Ann. Bot., 2003, 92, 487–511.
26. M. R.-D. R. Millaleo, A. G. Ivanov, M. L. Mora and M. Alberdi, Soil Sci. Plant Nutr., 2010, 10, 470–481.
27. M. R. Broadley, P. J. White, J. P. Hammond, I. Zelko and A. Lux, New Phytol., 2007, 173, 677–702.
28. T. Murata, T. Akazawa and S. Fukuchi, Plant Physiol., 1968, 43, 1899–1905.
29. J. M. Guevremont, D. R. Strongin and M. A. A. Schoonen, Am. Mineral., 1988, 83, 1246–1255.
30. M. J. Borda, A. R. Elsetinow, M. A. Schoonen and D. R. Strongin, Astrobiology, 2001, 1, 283–288.
31. M. J. Borda, A. R. Elsetinow, D. R. Strongin and M. A. Schoonen, Geochim. Cosmochim. Acta, 2003, 67, 935–939.
32. W. Wang, Y. Qu, B. Yang, X. Liu and W. Su, Chemosphere, 2012, 86, 376–382.
33. M. R. Kriegman-King and M. Reinhard, Environ. Sci. Technol., 1994, 28, 692–700.
34. R. Weerasooriya and B. Dharmasena, Chemosphere, 2001, 42, 389–396.
35. W. Lee and B. Batchelor, Environ. Sci. Technol., 2002, 36, 5147–5154.
36. H. T. Pham, M. Kitsuneduka, J. Hara, K. Suto and C. Inoue, Environ. Sci. Technol., 2008, 42, 7470–7475.
37. H. T. Pham, K. Suto and C. Inoue, Environ. Sci. Technol., 2009, 43, 6744–6749.
38. S. Oh, P. C. Chiu and D. K. Cha, J. Hazard. Mater., 2008, 158, 652–655.
39. C. Gil-Lozano, E. Losa-Adams, A. F.- Davila and L. Gago-Duport, Beilstein J. Nanotechnol., 2014, 5, 855–864.
40. W. R. Brown, J. Biol. Chem., 1936, 113, 417–425.
41. Y. P. Jiang, F. Cheng, Y. H. Zhou, X. J. Xia, W. H. Mao, K. Shi, Z. Chen and J. Q. Yu, J. Zhejiang Univ., Sci., B, 2012, 13, 811–823.
42. G. Barba-Espín, J. A. Hernández and P. Diaz-Vivancos, Plant Signaling Behav., 2012, 7, 193–195.
43. G. W. Luther I ii, Geochim. Cosmochim. Acta, 1991, 55, 2839–2849.
44. A. Kumar, I. Y. Galaev and B. Mattiasson, Bioseparation, 1998, 7, 129–136.

---

Footnote

† Electronic supplementary information (ESI) available: The detailed data, synthesis and characterization of the cerium and graphene oxide nanoparticles are given in section S2. See DOI: 10.1039/c4ra06861k

---