2.1. Nano pyrite synthesis: Nano pyrite synthesis is a two-step reaction. In the first step, we synthesize fresh polysulfide. We take an aqueous sodium hydroxide solution (0.8 M, 100 ml) divided into two equal parts. In one part, we purged hydrogen sulfide gas until it turned yellow-green, then the remaining half of sodium hydroxide (0.8 M, 100 ml) and 0.04 moles of sulfur were added. The solution was stirred in the presence of hydrogen sulfide for the next two h at room temperature to obtain yellow-orange polysulfide suspension. The suspension was filtered and stored at 4˚C. For the second step, fresh aqueous ferric chloride (0.04 M, 100 ml) was prepared in a round bottom flask and maintained at a pH of 5.6 using a buffer of 91 ml sodium acetate (0.2 M, 100 ml) and 9 ml acetic acid (0.2 M, 100 ml). The flask was placed in an oil bath with continuous argon purging and stirred continuously for one h at room temperature. Then to which, we slowly add 15 ml of polysulfide. The oil bath temperature remains at 120˚C for two h, and a dark grey suspension will appear. Then, the oil bath temperature was switched to 180˚C for the next 10 minutes to obtain a grey-colored suspension. The nano pyrite suspension settles at the bottom of the flask. We wash the suspension with hydrochloric acid, toluene, and acetone. We store the nano pyrite particles in a moisture-free chamber (Srivastava et al. 2014a, b; Das et al. 2016a, b, 2018; Jangir et al. 2019c, a, 2020b; Jangir 2020; Jangir et al. 2020a; Jangir and Das 2021).
2.2. Nano pyrite characterization: Structural features of nano pyrite were observed using X-ray diffraction (XRD) and Scanning electron microscopy (SEM). XRD pattern was obtained to study the characteristic planes of nano pyrite crystal, for which a powdered sample was analyzed using X' Pert powder-PANalytical XRD. Sample for SEM and EDS was prepared by drop-casting a suspension of nano pyrite in methanol on a copper stub. The sample was gold coated and observed using FEI, Quanta 200 SEM. Fourier Transform Infrared Spectroscopy (FTIR) was performed to investigate the bonding characteristics of the nano pyrite particles. The sample was prepared by making a pellet of nano pyrite powdered sample with KBr. Perkin-Elmer FTIR spectrum BX spectrometer was employed to obtain the FTIR spectrum. X-ray Photoelectron Spectroscopy (XPS) was performed using PHI 5000 Versa Prob II, FEI for powdered samples. XPS was used to study the surface characteristics of nano pyrite particles (Srivastava et al. 2014a, b; Das et al. 2016a, b, 2018; Jangir et al. 2019c, a, 2020b; Jangir 2020; Jangir et al. 2020a).
2.3. Field trial: Land preparation: We conducted the field trial in the institute nursery of the Indian Institute of Technology Kanpur, India. We selected a nutritionally deficient patch of land adjacent to the nursery’s greenhouse. We first plowed the ground and prepared three plots. Out of the three plots, we maintained the exact dimensions (3*4 ft2) for two plots, while for the third plot, we doubled the length and breadth of the plot (6*8 ft2). We use the third plot (6*8 ft2) as a nursery to grow the onion seedlings before we perform transplantation. For both seasons, the same protocol was used. In figure 1, we have presented a flow chart of the cropping protocol followed during the two years of field trials.
The nutrient profile of the soil: At the beginning of the field trial, we perform a soil nutrient analysis for Nitrogen (N), Phosphorus (P), and Potassium (K). N was estimated using the Kjeldahl method, and P and K were calculated using ICPMS analysis as described earlier (Jangir et al. 2019b, a).
Sowing, root treatment, transplantation, and manure application: For the season I, the field trial was initiated on November 10, 2018. 50 mg onion seeds were germinated in nursery beds (6*8 ft2). After thirty-three days (December 13, 2018), the germinated seedlings were harvested and were randomly divided into two sets of 100 seedlings each. Next, the root treatment was performed for three h before transplantation. In the control group, the roots of seedlings were soaked in water, while in the test group, the roots were soaked in 100 µg/ml aqueous suspension of nano pyrite. After three h of root treatment, the control and test seedlings were transplanted in their respective assigned plots of individual dimensions of 3*4 ft2. Goat droppings were used for manuring both plots (1 kg/plot) on January 19, 2019. The second (season II) cropping season follows a similar calendar.
Crop care: Recommended irrigations were given, and all other parameters were kept the same for both sets. All the other agronomic practices were followed as per the recommendations (Gupta 2019; Choudhury 2020).
Harvest: Harvesting was done manually, and the mud was removed and cleaned before weighing the samples.
2.4. Crop Analysis and growth Parameter: The crop was harvested and analyzed for plant growth parameters such as fresh weight, dry weight, leaf area, specific leaf area, leaf area index, size/area of bulb, and onion bulb weight, etc. The relative anthocyanin and flavonol content was also measured for control and test samples. The anthocyanin content of the bulb is an indicator of the coloration of the bulb and thus marketability.
The onion crop yield obtained at harvest was weighed for both control and test groups. Fresh weight was measured for both the whole plant (leaves and bulb) and bulb. The average yield obtained is reported in grams for bulbs and leaves. The weight of leaves was calculated by subtracting the weight of the bulbs from the weight of the whole plant. Total onion bulbs obtained from control and test groups were counted. Twelve and thirteen representative plants were chosen from the control and test groups, respectively. These plants were placed alongside a ruler, and a snapshot was taken. This picture was used to calculate the area of leaves and bulbs using ImageJ. Leaf area is a direct measurement of photosynthetic activity and transpiration. Standard errors have been reported for image analysis. The Student's t-test was performed to calculate the significance of the results. Leaves and bulbs were kept at 70 degrees Celsius for 6 hours in order to measure the dry weight of the samples. Once we had primary data, other plant growth parameters were calculated as per standard procedure using the following (Sudhakar et al. 2016):
Leaf Area Ratio: It is a measure of the relative size of the assimilatory apparatus. In simpler terms gives an idea of the portion of the plant involved in the photosynthetic activity.
Leaf Area Ratio = Total Leaf Area/Total Dry weight
Specific Leaf Area (SLA): SLA signifies the leaf density of the plant. It tells us about the amount of leaf area used to produce unit biomass.
Specific Leaf Area = Total Leaf Area/Leaf Dry Mass
Leaf Area Index (LAI): The leaf area index (LAI) of a plant is defined as its leaf area per unit of ground area. The leaf area index (LAI) is one of the most important parameters in plant ecology. It signifies how much foliage (leaf) is present on the plant. It measures the active photosynthetic area, as well as the area available for transpiration. More photosynthetic area signifies that the rate of photosynthesis and food production and accumulation is more. In comparison, higher transpiration rates ensure higher uptake of water and minerals. These two factors ensure better nutrition accumulation in edible parts of the plant.
Leaf Area Index = Total Leaf Area/Total Land Area.
The harvested plant materials were further tested for their anthocyanin and flavonol content in both leaves and bulbs. Flavonol is a class of flavonoids that participate in stress responses. These are the most primitive and widespread flavonoids and have a wide range of plant physiological functions. Anthocyanin is a specific class of plant pigments that belong to flavonoids. These pigments provide bright color to the onion bulb and thus increase the marketability and visual acceptability of the crop produce. The higher content of anthocyanin in onions bulbs also signifies earlier maturity, thus helping the farmers get a good price for their early produce. Anthocyanin is also a well-known antioxidant; thus a higher anthocyanin content ensures better health (Martín 2017).
Preparation of plant samples: Plant samples were finely cut into small pieces and homogenized using a pestle and mortar. 2 gm of this homogenized sample was mixed with 7ml of 80% ethanol (0.1% HCl) and further homogenized. The extract was filtered to obtain an alcoholic extract.
Flavonol measurement: Relative flavonol content was measured for control and test samples (leaves and bulb) by taking absorbance at 510nm.
Anthocyanin measurement: Relative anthocyanin content was measured using the ethanol extract and mixing in equal quantity with buffer (pH 4.5) and taking absorbance at 525nm.