Effect of Carbon Nanoparticles in Biochar and Sulphur as a Foliar Spray on Onion Plants: A New Orientation

Biochar contains raw carbon nanoparticles. Water-soluble dried carbon powder nanoparticles (wsCNPs) isolated from leftover biomass (waste stems and roots) in agricultural land can enhance the overall growth rate of onion (Allium cepa L.) plants. Field experiments conducted in 2017–2018 and 2018–2019 aimed to evaluate the effect of wsCNPs and sulfur as a foliar spray on vegetative growth, yield, and some nutritional values of onion plants. The biochar used in the study solution, namely, wsCNPs, was applied at 300, 400, and 500 mg/L incorporated with three levels of 2.5, 3.5, and 4.5 g/L of sulfur (Thiovit, 80%) as a foliar spray. Results indicated that treated plants with all levels of wsCNPs and sulfur had significantly higher values of vegetative growth parameters, photosynthetic pigments, yield, bulb quality, and phytochemical compounds compared with untreated plants. Using the highest foliar spray level of wsCNPs and sulfur obtained significant results for all evaluated characteristics among other levels. This increment was more pronounced when wsCNPs and sulfur were combined, indicating that spraying is a useful agriculture and eco-friendly practice for sustainable crop production.


Introduction
Onion (Allium cepa L.) is an important vegetable crop grown in Egypt for local consumption and exportation. It ranks third among the most important vegetable crops after tomato and potato in Egypt, which was the 9th biggest onion producer in the world in 2017. Its onion production area is 68,057 ha with a total production of 2,379,035 tons and average yield of approximately 36 t ha -1 (FAOSTAT 2017). Onion has high economic importance because of its taste and health-supporting qualities, including anticancer properties, antithrombotic and antiasthmatic activities, and antibiotic effects (Suleria et al. 2015). Egyptian onion varieties are of a high quality because of its high nutritional value and pungency. Therefore, it has a high potential for exportation. Globally, major efforts have been made to S ensure more and the best food production to bridge the food gap between production and consumption and to meet food demand of rising populations in developing countries. In achieving high yields, farmers apply nontraditional treatments such as the use of sulfur and biochar as foliar application.
At present, biochar is widely applied to agricultural soils as a soil conditioner. Biochar is traditionally used for growing plants because of its nutrient retention capability for need-based slow release (Zeeshan et al. 2014;Kumuduni et al. 2020). However, in this study, biochar was used as a foliar fertilizer for onion plants. Biochar contains raw carbon nanoparticles (Saxena et al. 2014). Carbon is a key element involved in life. The presence of carbon in organic compounds plays a role in biochemical processes, and elemental carbon in its newer nano version, including coal and graphite, has shown great importance in several applications (Jariwala et al. 2013). The use of any fertilizer in nanoform releases the nutrients at a slower rate for a longer period, thereby limiting nutrient loss and reducing pollution (Mahmoud et al. 2018). Biochar is carbonized from an organic material derived from organic biomass under specific conditions such as defined temperature, time, and oxygen-limited environment (Rizwan et al. 2016;Ali et al. 2017). In addition, biochar has unique characteristics such as high surface area, cation exchange capacity, porosity, pH, and functional groups, making biochar an efficient material, which can be used for plant growth (O'Connor et al. 2018;Ali et al. 2019). Abd Elwahed et al. (2019) stated that the content of photosynthetic pigments (chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids) of wheat plant was affected by biochar used as foliar fertilizer.
Onion is a sulfur-rich plant; sulfur is required for proper growth and yield (Mishu et al. 2013). Sulfur is an essential element for plant growth because it contains major metabolic compounds such as amino acids (methionine and cysteine), glutathione, sulfolipids, and proteins (Hussain and Leitch 2008;Shah Khalid et al. 2016;Shaheen et al. 2016). Sulfur not only increases onion bulb's yield but also improves its quality, particularly pungency and flavors (Jaggi and Dixit 1999). The yield potential of onion has not been fully exploited as sulfur fertilizer is used in very low quantity despite its very high requirement (De Souza et al. 2015;Chattoo et al. 2019). Apart from improving the yield, pungency, flavors, and other quality parameters, sulfur application improves bulbs' shelf life by providing resistance against pests and diseases (Magray et al. 2017). Hussain and Leitch (2008) applied sulfur as foliar application on wheat, which resulted in a significant effect compared with its application as a soil conditioner.
This study aimed to evaluate the use of biochar and sulfur as foliar fertilizer instead of their traditional use as soil conditioners and their effect on onion plants.

Site Description and Plant Material
Two field experiments were conducted during two successive growth winter seasons of 2017-2018 and 2018-2019 at a private farm in Quesna, EL-Menofyia Governorate, Egypt (latitude 30°33 0 38 00 N, longitude 31°00 0 29 00 E and altitude of 11 m above sea level). The physical and chemical properties of the experimental soil are shown in Table 1.

Synthesis of Water-soluble Carbon Nanoparticles
We collected a biochar material produced by the carbonization of plant waste left in agriculture fields used in the present study. The collected biochar was powdered, and the fine powdered part was separated from the unburned parts by screening. Screening was performed by adding water to the powdered material, and then the mixture was stirred. The heavier part containing silica and other solid inorganic materials settled down fast, leaving the upper part containing floating carbon particles, which were decanted off. Flotation was repeated, which resulted in the separation of essential carbon materials in almost pure form from silica and mud. The carbon part was then filtered using a filter paper and washed two times with water to remove any residual soluble salts present therein. The carbon residue was then dried at 60°C for 5 h. This carbon is labeled as raw carbon nanoparticles (Sonkar et al. 2012;Saxena, et al. 2014).

Experimental Treatments
The final isolated and dried carbon powder was used in the study solution and labeled as water-soluble carbon powder nanoparticles (wsCNPs), which was applied at 0, 300, 400, and 500 mg/L. Sulfur (Thiovit, 80%) was obtained from AL-Goumhoria Co., Egypt, and applied at a rate of 0, 2.5, 3.5, and 4.5 g/L as a foliar spray. In addition, biochar (wsCNPs) was used individually at 300 mg/L and incorporated with sulfur at 300 mg/L of biochar and 2.5 g/L of sulfur (low level), 400 mg/L of biochar and 3.5 g/L of sulfur as (mid-level), and 500 mg/L of biochar and 4.5 g/L of sulfur (high level). All treatments were sprayed three times with a 10-day interval starting at 30 days after planting date. Spraying was performed in the morning using a hand-pressure sprayer.

Experimental Design
The experiments were set in a randomized complete block design with three replicates per treatment. The experimental treatments (five treatments) were randomly arranged within the block, and each block consisted of three plots. Onion seedlings cv. Giza 20 were transplanted in the second week of December in two seasons. Seedlings were planted on drip irrigation laterals that were 1 m apart and 25 m long with a 25-cm distance between drippers (standard 4 L/h discharge at 1.5 bar drippers). Three irrigation lines were used as a border among treatments and were excluded from the experiment to prevent interaction among the treatment plots. Four seedlings were planted 10 cm apart around each dripper.

Experimental Site Preparation and Cultivation
As for fertilization, 30 m 3 of organic manure per feddan and 75 units per feddan of calcium superphosphate (15% P2O5) were added during soil preparation. Nitrogen fertilizer was added in the form of ammonium sulfate (20.6 N/fed) at 150 units per fed (last N dose was 90 days after transplanting, approximately 60 days before lifting). Potassium sulfate (48% K2O) was applied at a rate of 96 units per feddan two times. The first dose (48 units) was given during the preparation of soil for planting, and the second dose (48 units) was given at the beginning of the formation of bulbs. Cultural practices and disease and pest control management were in accordance with the recommendations of the Egyptian Ministry of Agriculture.

Vegetative Growth
Random sample of ten plants from each plot were collected at 75 days after transplanting to measure the vegetative growth parameters such as plant height (cm), number of leaves, diameter of bulb and nick (cm), and fresh and dry weight of the whole plant.

Leaf Pigment Contents
Photosynthetic pigments, that is, chlorophyll a, b, and a + b and carotenoid contents, were determined in fresh leaves sampled at 75 days after planting date based on Moran (1982). Fresh leaf disks (500 mg) were immersed overnight in 10 mL of N, N-dimethyl formamide. The obtained extracts were measured at 470, 647, and 663 nm using a ultraviolet-visible (UV-Vis) spectrophotometer (T-60, PG instrument, Wibtoft Leicestershire, UK) for carotenoids, chlorophyll b, and chlorophyll a, respectively, N, N-dimethyl formamide was used as blank.

Bulb Characteristics
During harvest (when bulbs reach the variety normal bulb size and skin color, which is approximately 150 DAT), onions were lifted by hand. Immediately after lifting, onions were subjected to field curing on the ground under a shaded area for 10 days. After curing, bulbs were sorted, and the following variables were measured: The average bulb weight (g) was calculated by dividing the total yield of the plot area by the number of bulbs. Dry matter percentage (D.M.%) was obtained by oven drying bulb tissue at 70°C for 72 h, and then the tissue was weighed on the basis of the initial fresh weight, neck diameter (cm), bulb diameter (cm), and bulb length (cm).

Yield and Yield Components
After harvesting and bulb curing, the following variables were also measured. Total yield (kg/m 2 ) was calculated on the basis of the total yield for the experimental plot.

Bulb Chemical Quality Parameters
Another sample was taken to the lab in the Vegetable Research Department, NRC, to measure bulb quality characteristics. Bulb quality was evaluated by measuring total nitrogen using the micro Kjeldahl method in accordance with the procedures described by Cottenie et al. (1982). Phosphorus percentage was assayed on the basis of the modified colorimetric (molybdenum blue) method using a spectrophotometer (SPECTRONIC 20 D, Milton Roy Co., Ltd., USA) in accordance with the procedures described by Cottenie et al. (1982). Moreover, potassium percentage was measured using flame photometry (JENWAY, PFP-7, ELE Instrument Co., Ltd., UK) as described by Chapman and Pratt (1982). Sulfur content was determined via the modified colorimetric method using a spectrophotometer as described by Chapman and Pratt (1982).
Total soluble solids (TSS%) were determined using the Hanna Digital Refractometer Model HI96801. The total phenolic content was determined in absolute ethanolic extract using a Folin-Ciocalteu reagent as described by Stratil et al. (2006) and Mahmoud et al. (2019). The reaction mixture absorbance was read at 725 nm using a UV-Vis spectrophotometer. Gallic acid was used as the standard reference, and total phenolic content was expressed as milligram gallic acid equivalent per gram of dry weight tissue (mg GAE/g DW). Total flavonoid content was determined in absolute ethanolic extract using the aluminum chloride colorimetric method described by Chang et al. (2002) and Mahmoud et al. (2019). The absorbance of the reaction mixture was measured at 415 nm using a UV-Vis spectrophotometer. The total flavonoid content was expressed as milligram quercetin equivalent per gram of dry weight tissue (mg QCE/g DW).

Statistical Analysis
The obtained data were subjected to two-way analysis of variance using the Statistical Package for the Social Sciences (SPSS 2008 release 17.0 for Windows, SPSS Inc., Chicago, IL, USA). Values were expressed as an average of three measurements ± standard deviation. The least significant difference test was used to compare significant differences among treatment means at p ≤ 0.05 level of significance in accordance with the procedures reported by Snedecor and Cochran (1980). Table 2 shows that the highest concentration of wsCNPs and sulfur had significantly improved all evaluated parameters of vegetative growth in both studied seasons (p ≤ 0.05). By contrast, the highest foliar application of wsCNPs and sulfur obtained significant values of plant length, number of leaves/plants, neck diameter, fresh and dry weights of leaves, and bulbs compared with other treatments in both gowning seasons, followed insignificantly by the application of mid-level and low-level treatments. Therefore, these treatments remarkably promoted plant vegetative growth parameters relative to control treatment (without foliar spray). Control treatment significantly obtained the lowest values of all measured characteristics of plant vegetative growth of onion plant compared with the rest of the treatments in both study seasons. In general, all treatments with wsCNPs water-soluble carbon nanoparticles, S sulfur, LSD least significant difference wsCNPs and sulfur enhanced all measured characteristics of vegetative growth of onion plant compared with plants sprayed with tap water (as control).

Yield and Physical Properties of Bulb
Data presented in Table 3 indicated that the highest values of all determined parameters of yield and physical properties of bulb (bulb diameter, bulb length, and average bulb weight) were recorded when onion plants were treated with wsCNPs and sulfur at the highest level (500 mg/L wsCNPs + 4.5 g/L sulfur). The same trends were observed in both seasons of the study. On the contrary, the onion plants that did not receive any treatments showed significantly (p ≤ 0.05) low values of total yield and physical properties of bulb in both seasons. Therefore, the parameters of vegetative growth shown in Table 2 were highly related to the total onion yield and some physical properties of bulb shown in Table 3, which ultimately increased the total yield. The total yield (ton/fed) and average weight of bulb (g/bulb) were increased by up to 32.64-35.13% and 29.30-29.66% between the first and second seasons, respectively, when onion plants received foliar application at the highest level of wsCNPs and sulfur. Furthermore, the increment of the average weight of bulb of onion plants was primarily attributed to the increase in total yield per plant accompanied by this treatment.

Leaf Pigment Contents
The highest significant (p ≤ 0.05) values of onion leaf pigment contents (chlorophyll a, b, and a, b, and carotenoids) K wsCNPs water-soluble carbon nanoparticles, S sulfur, LSD least significant difference wsCNPs water-soluble carbon nanoparticles, S sulfur, LSD least significant difference in both seasons caused by wsCNP treatment were combined with a sulfur mixture, whereas the control treatment obtained the lowest values (Table 4).

Nutritional Values of Bulbs and Their Mineral Contents
The phytochemical compounds, that is, total phenolics, total flavonoids, ascorbic acid, and TSS, and mineral contents in bulbs were higher with foliar application of a higher rate of wsCNPs and sulfur than other treatments in both growing seasons (Tables 5 and 6).
Foliar spraying treatments of wsCNPs and sulfur showed remarkable effects on bulb mineral contents, including N, P, K, Ca, and S, in both study seasons (Table 6).
A high level of wsCNPs and sulfur resulted in the highest significant values of mineral contents and phytochemical compounds in onion bulbs, followed by mid-level, lowlevel, and control treatments, which resulted in the lowest values of some chemical components.

Discussion
Onion plant growth characteristics were significantly increased with the increase in wsCNPs and sulfur application levels. These enhancements were related to the ability of carbon to enhance photosynthetic enzymes and cell growth of different organs of onion plants. These results have been discussed by Sonkar et al. (2012), who found that biochar is a co-product of bioenergy production, which can contribute to carbon sequestration goals. In addition, biochar nutrient content variation can be attributed to different feed stocks and different pyrolysis conditions (Chan and Xu 2009;Blackwell et al. 2015). Increasing onion growth-re-lated attributes could be due to the role of sulfur in balanced nutrition and in physiological functions such as the synthesis of sulfur-containing amino acids and development of the used root system, thereby resulting in increased nutrient uptake and photosynthesis and improved growth (Dudhat et al. 2011;Tripathy et al. 2018;Chattoo et al. 2019). In addition, sulfur is an essential element for plant growth because it is present in major metabolic compounds such as amino acids (methionine and cysteine), glutathione, sulfolipids, and proteins (Shaheen et al. 2016). K mentioned that treated plants with a high level of biochar and sulfur as an independent factor showed a significant increase in yield and quality in various crops. The increment of yield with the application of biochar is related to carbon nanomaterials, and intravascular wood enhances the absorption of water transport through the channel process or adsorption. Biochar slowly releases these nutrients, which allows for normal healthy growth, thereby improving the yield and components of plants. Mishu et al. (2013) mentioned that onion yield potential has not been fully exploited as a sulfur fertilizer because it is used in very low quantity despite its very high requirement. Onion yield potential has been achieved through foliar fertilization. Sulfur, a protein-forming nutrient, is gradually being documented as the fourth major nutrient for plant growth after nitrogen, phosphorous, and potassium (Khalid et al. 2016). Therefore, the increment in plant yield may be explained as vigorous vegetative growth as previously mentioned.
Similar results were reported by Saxena et al. (2014) and Abd Elwahed et al. (2019) who stated that foliar application of biochar increased leaf pigment contents of sugar beet. On the contrary, the increase in biomass by sulfur nutrition can be explained by its role in ferridoxin, a Fe-S protein in chloroplast (Khalid et al. 2016), which enhanced synthesis and maintenance of chlorophyll contents and improved photosynthetic rate and assimilating efficiency.
The enhancement of biochar application on leaf pigment contents might be attributed to the delaying of leaf senescence by decreasing leaf pigment decomposition or improving its biosynthesis in spinach (Danish et al. 2019).
These results might be due to the high content of phenolic compounds and antioxidants in this mixture, which might affect metabolic processes, leading to the accumulation of phytochemical compounds and increasing antioxidant activity. Consequently, the nutritional values of the treated plants are enhanced. Furthermore, vegetables of the Alliaceae family tend to accumulate biologically active and strong antioxidant substances (Mishu et al. 2013). Therefore, the role of biochar is important to trap the anionic and cationic forms of nutrient ions, followed by their slow delivery as required by plant growth. In addition, biochar has the optimum capability to supply nutrients for plant growth (Saxena et al. 2014;Ali et al. 2019). Similarly, Ali et al. (2019) reported that the application of carbon nanotubes can significantly increase the carbon/nitrogen ratio in rice root, whereas nanocarbon application significantly inhibited nitrogen assimilation.
Increasing bulb sulfur content resulting from spraying of biochar and sulfur can affect bulb quality, particularly its pungency, which is defined as the combination of onion flavor and odor caused by the concentrations of sulfonic and thiosulfonic volatile acids containing sulfur (De Souza et al. 2015). Similarly, sulfur as a foliar fertilizer can affect bulb quality, particularly its pungency, which is defined as the combination of onion flavor and odor caused by the concentration of sulfonic and thiosulfonic volatile acids containing sulfur (De Souza et al. 2015). Sulfur not only increases the bulb yield of onion but also improves its quality, particularly pungency and flavor (Jaggi and Dixit 1999).

Conclusion
The abovementioned results indicated that wsCNPs incorporated with sulfur are considered as a useful agriculture and eco-friendly practice for sustainable crop production. In addition, remarkable values of vegetative growth, yield, and quality attributes of onion plants grown under drip irrigation system conditions can be obtained by foliar spraying 500 mg/L of dried wsCNPs combined with 4.5 g/L of sulfur (Thiovit, 80%) three times after 30 days from planting date for every 10 days.