Sustainable nano-interventions to enhance crop yield, anthocyanin content, and marketability of onion (Allium cepa)

Our reliance on synthetic agrochemicals has detrimental effects on the environment and living species. To restore the ecological balance, we need to explore strategies to minimize agrochemicals’ use without compromising agricultural production. Nano-formulations help lower dosages of agrochemicals, leading to the emergence of nano-agriculture. Judicious use of nanomaterials for enhancing plant metabolic performance and yield remains a challenge. Here, we have achieved the same by root treatment of the onion crop with nanopyrite (FeS 2 ) and further organic goat manure fertilizer application. We report an increase in yield through root treatment with nano-pyrite plus soil application of goat dropping (Test) as compared to the use of goat dropping alone (Control). The total biomass of the test sample was 4.75 kg (n=86), while control samples weighed 3.5 kg (n=86). The total bulb weight for the control and test was 2.6 kg and 3.6 kg, respectively. We observed a yield-boosting effect of root treatment with nanopyrite in onion. There is a significant increase in the anthocyanin content in test bulbs and flavonol content in test leaves. We have integrated two types of farming systems: organic and nano-agriculture to bolster the metabolic fitness of the onion ( Allium cepa ) and achieve sustainable food production.


Introduction
Through evolution, humankind has developed various strategies for food production. When we as a race shifted from food gatherers to food producers, the first form of farming emerged as natural farming, relying on minimal interference to the ecosystem 1-3 . Even today, many parts of the world follow natural agriculture. Wisdom-driven advancement of human skills led to the observation that organic matter in the soil helped improve crop produc-4 follow-up field trials on chickpea 27 , spinach 28 , beetroot, carrot, fenugreek, alfalfa, mustard, and sesamum 29 , we observed higher productivity following pyrite seed treatment. In pyrite-treated rice, an NPK equivalent yield was reported 30 . Jangir et al. recently showed an improved result in wheat crops following nano pyrite seed treatment 31 . The nano pyrite seed treatment gets nullified by co-treatment with an antioxidant nanomaterial, viz., cerium oxide 32 . Recently Jangir et al. discovered that upon treating the roots of chili, marigold, cabbage, cauliflower, and tomato in an aqueous suspension of nano pyrite for three hours, a significant increase in yield was observed [33][34][35][36] .
Further seed and root treatment with nano pyrite improve the plants' root foraging ability 27,31,34-37 .
The current study addresses the synergistic effect of nano pyrite root treatment and organic manure (goat droppings) on onion production (Allium cepa). It has tremendous commercial significance. It is a temperate crop, and India is the highest contributor to onion production after China. It has multiple uses-as a spice, salad, and vegetable. The allyl propyl disulfide contributes to onion pungency. The soil has to be rich in organic matter for optimal onion production. Recommended fertilizer dosage is 125:60:100 kg/ha of Nitrogen (N): Phosphorus (P): and Potassium (K) 38,39 .

Material and methods
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 26,27,37,28-32,34-36 .

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 26-32,34,35,37 .

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 ft 2 ) for two plots, while for the third plot, we doubled the length and breadth of the plot (6*8 ft 2 ).
We use the third plot (6*8 ft 2 ) as a nursery to grow the onion seedlings before we perform transplantation. For both seasons, the same protocol was used. All methods were performed in accordance with the relevant guidelines and regulation. 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 33,34 .
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 ft 2 ). 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 ft 2 . 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 38,39 .
Harvest: Harvesting was done manually, and the mud was removed and cleaned before weighing the samples.

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 40 : 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 41 .
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.

Nano pyrite synthesis:
Iron pyrite nanoparticles were synthesized as described in our previous work via a low-temperature nucleophilic reaction [26][27][28][29][30][31][32]34,35,37 . The reaction involves iron (III) chloride and sodium polysulfide, the chief intermediaries being FeS and FeSH+. These intermediates are further attacked by polysulfide nucleophiles, which leads to the formation of FeS2. The reaction was carried out in an inert environment to prevent interaction with oxygen.   Figures 3a and 3b show the EDS analysis. Figure 3a shows the EDS overlay with the SEM image, and figure 3b shows the EDS spectrum for nano iron pyrite. FTIR peaks at 400-800 cm-1 marked pyrite bonding characteristics, the peak at 1076.5 cm-1 showed S-O bonding characteristics marking surface oxidation, and the presence of moisture was marked by peaks at 1623.8 cm-1 and 3407.6 cm-1 (Figure 4).

Field trial:
Nutrient profile of the soil: In the present study, a nutritionally deficient patch of land (total nitrogen (159 kg/ha), total phosphorus (0.3 kg/ha), total potassium (0.4 kg/ha)) was selected in the Institute Nursery of Indian Institute of Technology Kanpur, India. In earlier work, it has been shown that the above-mentioned nitrogen, phosphorous, and potassium level is considered nutritionally deficient 31,33,34 .   7a). In order to investigate the distribution of the weight between foliage and the economic yield, the leaves and the bulbs were weighed separately. The average bulb weight for test samples was significantly higher (41.86 gm) as compared to control (31.32 gm) (Figure 7e). Similarly, the average fresh weight of test leaves (13.37 gm) was higher in comparison to control leaves (10.84 gm) (Figure 7c).

Crop Analysis
Average water accumulation in both control and test plants was approximately equal. Since the fresh weight of the test samples was high, the average dry weight of the test plants (8.13 gm) was also more as compared to the control (6.158) untreated plants (Figure 7b). An approximately equal amount of moister content was found in both test and control sample leaves and bulbs. The average dry weight of control leaves was found to be 0.889 gm while the test leaves had an average dry weight of 1.096 gm (Figure 7d). Similar to the leaves the bulbs also accumulated the approximately same amount of water for both control and test samples. Therefore, the average dry weight of test bulbs (7.04 gm) was higher as compared to the control onion bulb samples (5.26 gm) (Figure   7f). 14 The average bulb area for test samples (2.33 cm 2 ) was significantly higher as compared to control samples (1.59 cm 2 ) (Figure 8a). Significantly denser leaf foliage was observed in test samples with an average leaf area of (11.53 cm 2 ) as compared to control (6.99 cm 2 ) (Figure 8b). The increase in leaf area resulted in significantly higher specific leaf area (10.51) (Figure 8c), leaf area index (0.08) (Figure 8d), and leaf area ratio (Figure 8e) in test samples as compared to control (7.86, 0.05, and respectively).  The relative anthocyanin content in control leaves (0.052) was considerably higher as compared to test leaves (0.03) (Figure 9a). However, there is a highly significant increase in the anthocyanin content in test bulbs (0.069) as compared to control bulbs (0.02) (Figure 9b). The results indicate that there is a selective partitioning of the pigment in leaves and bulbs. The test plants tend to accumulate significantly higher amounts of pigment in the food storage/edible parts. The relative flavonol content in test leaves (0.253) (Figure 9c) was found significantly higher with respect to control samples (0.086). On the other hand, the flavonol content in the bulb (Figure 9d) was approximately equal in both control and test samples.

Discussion
As mentioned earlier that philosophically, this work is inspired by an extreme ecosystem viz., hydrothermal vents in the deep ocean rifts of the pacific, where microbes like archeobacteria and thiobacillus receive their energy from nano pyrite and other metal sulfides. The fundamental hypothesis for the use of nano pyrite pivots around an exothermic process, whereby metal sulfides are converted to sulfates to liberate 200 kcal/mol of energy 42 . This kinetics is further accelerated when metal sulfides are covered with a thin film of water approximately 4% of their weight in the presence of oxygen 42 . Subsequent reduction of oxygen leads to the release of trace amounts of H2O2, which further attacks additional sulfide molecules resulting in the generation of Fe 2+ , Fe 3+ , SO4 2-, and S 0 . So, when plant roots are treated in aqueous suspension of pyrite, it is briefly exposed to iron, sulfur, pyrite, peroxide, and proton-rich environment. These species orchestrate a cascade of metabolic changes that enhance plant metabolism [33][34][35][36] . In other words, this treatment results in metabolically smarter plants. Here, we tested the feasibility of this root treatment in plants with shallow root system to draw nutrients efficiently from organic matter. The overall premise of the work is summarized in figure 10. The experiment was purposefully conducted in a nutritionally deficient patch of land to study the dual effect of root treatment and organic manure, thus, closely simulating a sustainable organic farm environment with a nano intervention. We chose goat dropping as an organic supplement as it has high contents of organic matter, nitrogen, and phosphorus, and indirectly impacts fertilizer mobilization and biological cycling. Also, the total nitrogen mineralization over one-twenty days has been found maximum for goat droppings as compared to that of poultry and cattle [43][44][45][46] . Further, goat manure is commonly used as a source of liquid fertilizer 47 .
Earlier, it has been observed that organic manure solely cannot suffice for maximum onion productivity. This could be due to the shallow root system of onion crops with lesser root penetrability. Thereby, onion crop requires nutrient application close of the root system, which was the principal reason that we applied goat manure on the soil surface [43][44][45][46] . Here, by pre-treating onion seedlings with nano pyrite before manure application, we demonstrated that boosting the metabolism of onion seedlings in the test group lead to better nutrient acquisition and higher yield as compared to the control group. Higher leaf area, specific leaf area, leaf area ratio, and leaf area index indicate a higher photosynthetic rate and higher transpiration. All these factors may have contributed to an increase in the size of onion bulbs of test plants. Bigger bulbs ensure better market and price for the farmer.
An increase in anthocyanin content in bulbs indicates that the economic yield is brighter colored and has matured earlier. It also indicates that the test yield shall have better marketability and may sell at a higher price. Higher flavonol content in test leaves ensures better stress response, particularly to UV-B radiation 48 . Flavonols play very important role in pollen germination and thus plant fertility 49 . The results indicate that the test plants have significantly higher flavonols and thus are less prone to UV stress, also; the plants may have higher fertility and therefore higher seed production capability.
In summary, on treatment with an aqueous suspension of nano pyrite, the root experiences a brief energy-rich microenvironment, which shapes its subsequent metabolism and growth. These metabolically boosted plants acquire nutrients efficiently from organically supplemented soil and hence have higher competitive fitness. This approach has the feasibility to open up avenues for nano-organic sustainable farming and further boosting the economy of goat farmers of Asian and African continents.
While sustainable farming aims at reducing chemical fertilizer by using organic manure, bio-fertilizers, nano fertilizers; a crop-specific increase in metabolic fitness could positively influence the yield. Here we show that a nano pyrite root treatment during transplantation could boost the metabolic fitness of the plant so that it can capitalize on the maximum benefits derived from organic farming; thus leading to a novel nano-organic farming strategy.       Average total dry weight in grams. c. Average leaf fresh weight. d. Average leaf dry weight. e. Average bulb fresh weight. f. Average bulb dry weight. Vertical bars denote 5% error bars.