Nano-Fertilization of Phosphorous and Potassium, Spraying Sepehr 4 Nano-Fertilizer and Carbon Nanotubes on some Qualitative Traits and Active Substances in the Grains of Rice (Oryza sativa L.)

The study aimed to investigate the response of rice crop (Oryza satival L.), local variety Anbar 33, to nano-phosphorus and nano-potassium, spraying with carbon nanotubes and synthetic nano fertilizer Sepehr 4 (K+Fe+Zn+Mn). Some indicators of rice grains traits were used to evaluate the response. The field experiment was conducted in Al-Taliaah district 41-Al-Husseiniyah in the southern pater of Babylon Governorate - Iraq. The experiment was carried out according to the Randomized Complete Blocks Design (RCBD), and the treatments were randomly distributed according to a straightforward one-way experiment. Phosphorus nano fertilizer at two levels 0 and 10 kg ha-1 and potassium nano fertilizer at two levels 0 and 10 kg ha-1, and the treatments of nanomaterials of carbon nanotubes 50 mg L-1 and Sepehr 4 fertilizer (K+Fe)+Zn+Mn, 2.5 ml L-1 it was sprayed on the shoots of plants. The applications were conducted four times of nanomaterials with combinations of foliar sprays and ground additives to the soil, so that the number of treatments is sixteen, with three repetitions. The results of the gas chromatograph coupled with mass spectrometry (GC-MS) presented that the treatment of N-K + (CNT) + Nano(K + Fe + Zn + Mn) potassium nano fertilizer, carbon nanotubes spraying and Sepehr 4 fertilizer K + Fe + Zn + Mn resulted in the highest percentage of the saturated fatty acid content of rice grains 2.16% and volatile oils 6.82%. The treatment of N-(P+K)+N-(K+Fe+Zn+Mn) phosphorus and potassium nano fertilizer and Sepehr 4 fertilizer resulted in the highest value in the content of palmitic acid, 26.41% in rice grains. The treatment of N-(P+K)+(CNT) +N-(IIII) phosphorus and potassium nano fertilizer and spraying carbon nanotubes fertilizer and Sepehr 4 fertilizer resulted in the highest content of rice grains of unsaturated oleic acid, 3.95%. N-K+ (CNT) Potassium nano fertilizer and carbon nanotube fertilizer spraying resulted in the highest value of rice grains’ unsaturated linoleic acid content, 6.94%.


Introduction
One of the benefits of nanotechnology is its rapid absorption, transport, and representation within plant tissues, as well as its stability under varying environmental conditions.It stimulates growth and productivity in quantity and quality.They are less depleted than traditional mineral fertilizers [1].Their particles have dimensions of 1-100 nanometers, which makes them have a high surface area with slow release and smart targeting according to the plant's needs.Its proper scientific use in the agricultural field works to reduce the loss of fertilizer nutrients, sedimentation in the soil, and environmental pollution.The fertilizers manufactured according to nanotechnology work very efficiently because their small quantities are added [2,3].Nano phosphorus has a role in disease resistance, stem strengthening, flowering, seed formation and development, root length and density, root hairs, and resistance to braking [4].Nano-potassium is an immobile element that moves via diffusion and mass flow [5]; it is a catalyst for over 60 enzymatic reactions and the synthesis of adenosine triphosphate (ATP).Potassium plays a role in the stomata's opening and closing [6].Foliar feeding is one of the means that has a role in meeting the plant's needs for nutrients in the leaves.The stomata in the leaves or the cell walls and membranes of the plant through which the nutrient solutions are absorbed when sprayed on the vegetative system at a specific concentration and at an appropriate time on the plant [7].Spraying the rice crop with nano-synthetic fertilizers Potassium K, Manganese Mn, Zinc Zn, and Iron Fe, which contains a quartet of nutrients, including potassium, on the leaves, is more efficient than the fertilizers applied to the soil.As a result, it increases grain productivity [8,9].Zinc contributes to the formation and maintenance of plant cell membranes [10].Manganese does not participate in chlorophyll production, but it has a role in its composition and is associated with the activity of 35 enzymes [11].Because iron has a role in the formation of ferredoxin and cytochromes that have a role in the process of photosynthesis and respiration and participates in the formation of protein due to nitrate reduction [12].The spraying of carbon nanotubes (CNTs) fertilizer on rice crops is considered one of the crucial nanomaterials that have received attention for their physical, chemical, and electronic properties [13].They are classified into single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) [14,15].The researchers noted that the multi-walled carbon nanotubes have a role in seed germination and seedling growth, as they improve the absorption of water and nutrients, including calcium and iron, and thus improve seedling growth and seed germination [16].Plant metabolites, including proteins, carbohydrates, and metabolites, are classified as a storehouse of natural substances.The active substances have different chemical structures and groups: enzymes, alkaline, phenols, steroids, amines, and glycosides [17].Numerous studies have proven the presence of more than 500 active substances in rice grains [18,19].Fixed oils are esters of fatty acids united with glycerol and stored in the seeds.They are among the vital compounds insoluble in aqueous solutions, but they dissolve in non-polar organic solvents and do not evaporate or volatilize.Volatile oils give plants their pleasing aroma and are organic compounds derived from primary plant secretions [17].Gas Chromatography / Mass Spectroscopy (GC-MS) is gas chromatography (GC) along with mass spectrometry (MS) used to separate different substances within the sample and determine their types and proportions [18,19].Rice (Oryza sativa L.) is one of the cereal commodities that ranks second in the world, after wheat, as a sustenance and economic resource for the Asian continent [20].The production and consumption of the Asia-Pacific regions account for more than 90% of the world's rice production [21].Rice is a cereal crop grown in 114 countries in the world, i.e.; it is found in a wide range of climatic zones extending from tropical countries to warm temperate and subtropical countries up to 40m south latitude and 50m north of the equator [22].Therefore, this study intended to understand the effect of nano-fertilization with phosphorus and potassium, spraying nano synthesis fertilizer (N, K, Fe, Zn, Mn ) and carbon nanotubes, single and combined, on the active substances of rice grains of the local variety Anbar 33.

Experiment Site
The field study was conducted in Al-Taliaah district 41-Al-Husseiniyah in the southern pater of Babylon Governorate -Iraq, within latitude (N-3560050) and longitude (E-481907), for the period from June 15 th, 2022, to November 19 th, 2022, which is the period required for the growth of the rice crop.

Experiment Design
The experimental interventions were randomized according to a simple one-way experiment, and the experiment was conducted using the Randomized Complete Blocks Design (RCBD) with three replications.Each sector contains sixteen plots.According to the proposed plan, phosphorous nanofertilizers were applied at 0 and 10 kg ha -1, and potassium nano-fertilizer at 0 and 10 kg ha -1 .In addition, spraying treatments were applied with Sepehr 4 nano-synthetic fertilizer (N,K, Fe, Zn, Mn) at a concentration of 2.5 ml L -1 and carbon nanotubes at 50 mg L -1 .Four-time sprays bring the experimental units to 48 [26].
Table 1.Fertilizers used in the experiment and their components.

Field Preparations
Cross-plowing, smoothing, and leveling of the field soil were carried out.A sample of the field soil was taken at a depth of 20 cm from five locations, represented by the four sides of the field and the middle, to be a representative sample of the field soil.The cultivation soil was characterized as sedimentary soil with a silty clay loam texture and classified as Entisols according to the modern American classification [23].The soil sample's laboratory analyses were conducted following the methods reported by [14].These examinations included electrical conductivity (EC) = 2.2 dS cm -1 , degree of reaction (pH) = 7,40, available nitrogen = 20.55 mg N kg -1 , available phosphorus = 13.10 mg P kg -1 , available potassium = 200.20 mg K kg -1 , and organic matter = 12.30 g kg -1 soil.The experimental field area was 300 square meters divided into three equal sectors.Each sector was divided into 16 plots, each of which dynameters was 3 x 1.5 = 4.5 square meters.The necessary ducts were opened to irrigate the plots, and the culverts were needed to drain the excess water.

Implantation and Crop Service
Rice seeds of the local variety Amber 33 certified by the Rice Research Station in Al-Mishkhab District -Al-Najaf Governorate were sown on June 15 th, 2022.Sowing was carried out at the rate of 120 kg seeds per ha -1 .First, seeds were sown directly by scattering them on the previously prepared soil and then covered with soil to prevent them from being washed away with irrigation water and picked up by birds.After that, the soil was irrigated, and the irrigation process continued every 3 days, from germination irrigation until grafting.The depth of the irrigation water was maintained at about 10 cm to ensure the availability of the appropriate amount of water required for plant growth up to the stage of full maturity of the plants.Irrigation water was cut off from the field 15 days before harvesting, and the weeds were removed continuously.

Fertilization
Nano phosphorus and Nano potassium fertilizers was applied in 0 and 10 kg ha -1 for each type of fertilizer, which was applied to the soil before sowing, Table (2).The applied quantities were calculated according to the area of the plots.

Foliar Fertilization (Spraying)
The nanomaterials, including carbon nanotubes, were sprayed at 50 mg L -1 and Sepehr 4 quadruple nano-synthetic fertilizer(N+K+Fe+Zn+Mn) at a concentration of 2.5 mg L-1 for each time.Four times of each nanomaterial was applied before the flowering stage and at the 50% stage of flowering, with a difference of 14 days between each time, according to the fertilizer manufacturer's instructions, by foliar spraying on the shoots of the plant (400 liters ha-1), Table (2).

Harvest
The harvest occurred on November 26 th, 2022, when all the plants had reached full maturity, and the grain moisture content ranged between 18-25% [24].

The Studied Traits
Content of rice grains of active substances using gas chromatography-mass spectrometry.Fife grams of rice powder was taken from each treatment as per the method of [25], and 5 ml of 50% ethanol was added to it with continuous stirring.Then filtration using filter papers, and then the filtrate was taken.As for the sediment, it was extracted with ethanol in the same way.The second filtrate was collected with the first, concentrated at 40 °C and kept at 4 °C for 24 hours.Then, ten milliliters of hexane was applied, 10 ml of chloroform was applied and dried, and the dry substance was dissolved in 5 ml of 20% methanol.A single milliliter of the resulting extract and injected 2 microliters of it into the GCMS-QP2010 Ultra, which includes the AOC-20i automatic identification unit for compounds based on the mass spectra according to the following conditions:  The separation column consists of 100% dimethyl polysiloxane with dimensions (0.25nm×30×1µmdf)  Helium carrier with a flow rate of 1 ml min -1  The temperature of the injector is 250 ºC, and the temperature of the ionic source is 200 ºC.
The oven temperature is automatically programmed for a temperature gradient.It starts from 40 ºC (equal temperature for 3 minutes) and increases by 15 ºC every minute to 180 ºC and then increases by 10 ºC every 3 minutes to 300 ºC, after which the temperature stabilizes at 300 ºC. The total time for each sample is 28 minutes. The mass spectra were taken based on 70 eV, with a time interval of 0.5 seconds and a fission rate of 40 to 450 Daltons.Components were identified using the National Institute of Standards and Technology (NIST) database by comparing the resulting spectrum of the unknown component with known stored components in the NIST library.Then, depending on the importance and the highest percentage, the active compounds were selected: Palmitic Acid, Stearic Acid, Oleic Acid, Linoleic Acid, and Volatile Oil.

Statistical Analysis
The experimental data were statistically analyzed, and the differences between the treatments in the RCBD design were calculated at a significant level of 0.05 using L.S.D test [13,26].

Fixed Oils: Saturated Fatty Acids (Stearic Acid and Palmitic Acid)
The Figure (1) indicates that the treatment N-(P+K) + N-(K+Fe+Zn+Mn) resulted in the highest percentage of palmitic acid 26.41% in the content of rice grains, compared to the lowest percentage when Nano-K fertilizer N-K treatment 15.09 %.However, the percentage of the control of stearic acid was 22.24%.Compared to its benchmarks, an increase of 4.17% in saturated fatty acid palmitic acid was achieved.This increase in palmitic acid content in rice grains is significant.This indicates, in general, the stimulating effect of nano-fertilizers added potassium, nano-phosphorus, and quaternary fertilizer spraying N-(K + Fe + Zn + Mn) in improving the nutritional content of rice grains [18,19].Shows the Figure( 2) the treatment N-K + (CNT) + Nano (K + Fe + Zn + Mn) resulted in the highest percentage of stearic acid, 2.16% in rice grain content, compared with the lowest percentage when treating Nano-P fertilizer N-P 0.83%, and the percentage of the control of acid 1.15%.With this parameter, the percentage of saturated fatty acid, stearic acid, increased by 1.01% compared to the control.This increase in stearic acid content in rice grains is considered to support the nutritional content [27,28].
Table 3. Saturated and unsaturated fatty acids and volatile oils in grains of rice.

Unsaturated Fatty Acids
The Figure (3) indicates that the treatment N-(P+K) + (CNT) +N-(IIII) resulted in the highest grain content value of unsaturated fatty acid oleic acid 3.95% compared to the lowest one with (N-) treatment (P+K) + spray (CNT) (0.48) %, while the percentage for the control was 1.04%.This is due to the stimulatory effect of producing unsaturated fatty acids from saturated fatty acids in alpha oxidation reactions [18].The N-K + Spray (CNT) treatment in Figure (4) resulted in the highest level of grain content of the unsaturated fatty acid linoleic acid 6.94% compared to the lowest level of Nano-P fertilizer (N-P), 3.04%, while its percentage of the control was (3.05) %.The increase in oleic acid resulted from the catalytic action of unsaturated fatty acid production reactions, which may explain the abiotic tension Treatments that occurred on the rice plant to spraying and nanomaterials.the plant's reaction was to produce antioxidant compounds, including unsaturated fatty acids.It is a way for the plant to defend itself to complete its life cycle.It is worth noting that the difference in the production of linoleic acid was double the production of the quantity, which is an improvement in the nutritional content of rice grains [28,29].

Volatile Oils
The Figure (5) indicated that the treatment N-K + (CNT) + Nano(K + Fe + Zn + Mn) resulted in the highest content of volatile oils, 6.82%, compared to the lowest level of the +N-treatment (K + Fe + Zn (+Mn) (N-P+ (CNT) 1.50 %, and the ratio in control was 2.65%.These results are consistent with the tests conducted on the grains produced with the experimental treatments, which supports this and improves the qualitative characteristics that increase consumer demand for rice grains to be distinguished by their aroma [17,30].

Conclusions
The most significant result of this study is the highest averages of the specific characteristics of rice grains as a result of the treatment with N-K + (CNT) + Nano (K + Fe + Zn + Mn) nano-fertilizers, nano-potassium nano-fertilizers, spraying carbon tube fertilizers, and N-(K + Fe +Zn+Mn).The active substances increased the fixed oils, the saturated fatty acid citric acid, and the volatile oils in the rice grains of the local variety, Anbar 33.The combined effect of the N-(P+K)+N-(K+Fe+Zn+Mn) treatment of phosphorus and potassium nano fertilizer and the quaternary N-(K+Fe+Zn+Mn) fertilizer increased the content of saturated palmitic fatty acid in rice grains, mystic acid.The treatment N-(P + K) + (CNT) + N -(IIII) had a combined effect of each of the phosphorous and potassium nano fertilizers and carbon nanotubes fertilizer spraying and the quaternary fertilizer N -(K + Fe + Zn + Mn) and increased the content of rice grains from the unsaturated fatty acid oleic acid.The treatment of N-K + Spray (CNT) potassium nano fertilizer and spraying carbon nanotube fertilizer increased rice grains' unsaturated fatty acid linoleic acid content.

Figure 2 .
Figure 2. Content percentage of Stearic saturated fatty acid in rice grains.

Figure 3 .
Figure 3. Content percentage of Oleic unsaturated fatty acid in rice grains.

Figure 4 .
Figure 4. Content percentage of Linoleic unsaturated fatty in rice grains.

Figure 5 .
Figure 5. Content percentage of volatile oils in rice grains.

Table 2 .
Fertilization experiment treatments and spraying of nanomaterials.

Spray Concentrations of mg or ml L -1 Treatments
Content percentage% of Palmitic saturated fatty acid in the rice grains.