Nanocitrates of iron (Fe) and zinc (Zn) are newer plant nutrients that can be employed in agriculture. Here, we examined the behaviour in soil and uptake of Fe and Zn nanocitrates by groundnut plants of Fe and Zn nanocitrates through soil application technique and compared it with the commercial counterpart. The combined nanocitrate compositions were also formulated by the physical mixing and characterized. The plant uptake of Fe and Zn was determined through atomic absorption spectrometry (AAS). All the treated plants had shown good germination and higher vigor index compared to the control treatments. The highest Fe and Zn content in soil and uptake was found in nanocitrates was followed by commercial fertilizers. Among 34 synthesized citrates, BFC (1:1)- 6; BZC (1:3)- 6; BFZ (4:6)- 8; BFZ (5:5)- 2; BFZ (5:5)- 6; and BFZ (8:2)- 4 are promising trends to evaluate under field conditions with higher stability.
Article
Fe and Zn metal nanocitrates as plant nutrients through soil application
https://doi.org/10.21203/rs.3.rs-1714946/v1
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iron
zinc
mineral nutrients
nanocitrate
incubation
leaching
soil availability
Nanoparticles (NPs) interact at the molecular level in living cells and employing nanoparticles in agriculture, expecting that these particles impart some beneficial effects in seed germination and control of plant diseases. Using NPs and nanopowders as carriers, controlled- or delayed-release fertilizers can be developed. NPs are highly reactive due to specific surface area, high-density reactive areas, or increased reactivity on the particle surfaces. Besides, the same features facilitate easy absorption of pesticides and fertilizers produced at the nanoscale1. Thus, nanotechnology can revolutionize agriculture with new tools to enhance the ability of plants to absorb nutrients. The broadcast, foliar spray, and pre-sowing seed treatment methods were used for applying micronutrients2,3,4. Furthermore, applying micronutrients through soil and seed treatment could assist the plant through the critical stages of early growth and significantly increase yield parameters.
Benefit of nanotechnology as nano-fertilizers and other nanomaterial-based designs have much more potential to boost agriculture5,6,7,8. In the recent past, using nanoparticles (NPs) in agriculture has become more popular, and NPs as fertilizers are very effective9,10,11,12. Thus, NPs can be used to control release of concerning nutrients, and it would be a helpful strategy to resolve the issue of low agronomic productivity13. Among various other NPs, zinc-oxide nanoparticles (ZnO-NPs) improved plant growth while abating oxidative damage in cotton14,15. Silicon nanoparticles (Si-NPs) enhanced plants' photosynthesis and biomass under stressful conditions16. Applying iron nanoparticles (Fe-NPs) improved plant development and reduced metals uptake in wheat plants under stress17. The mechanisms underlying NPs-mediated nutrient uptake vary with NPs type, mode of application, time of NPs exposure, and plant species. In this way, NPs can enhance plant improvement and nutrient uptake by minimizing nutrient losses or enhancing the plant defense system16,17,18.
Micronutrients play a vital role in plant nutrition and growth. Iron is one of the essential elements for plant growth and crucial in photosynthetic reactions, activating numerous enzymes involved in the transfer of energy, reduction and fixation of nitrogen, formation of lignin, and contribution in ribonucleic acid (RNA) synthesis19. Zinc (Zn) is the second most abundant transition metal in organisms after iron. Also, Zn is the only metal represented in all six enzyme classes: oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases20. Furthermore, several reactions are catalyzed by iron and zinc compounds in plants. Iron and zinc are essential micronutrients for humans, animals, and plants. According to Graham et al.21, throughout the world, more than 3 billion people suffer from micronutrient deficiencies. As a result, there is an increase in the research towards developing technologies for increased uptake and accumulation of micronutrients in comestible plant parts.
Numerous studies have reported applying a single type of NPs under biotic and abiotic stresses in various crops. Many of the studies have been conducted in pot experiments. Little is known about NPs role in leaching sedimentation studies under laboratory conditions. It was hypothesized that the combined application of various NPs could effectively synergistically positively affect plants. Both Fe and Zn are chosen due to their importance as plant nutrients and their deficiency in world soils. Metal NPs, including nanoscale Fe and Zn, may interact with SODs (super oxidase dismutase) and modify their biochemistry and functioning since Fe and Zn are structural constituents of SODs. The combined Fe-SOD and Zn-SOD seem to be the groups following the metal co-factor used by the plant22.
Chelated-based micronutrients finding its prominence due to protection from fixation and excess washout. Among available chelators EDTA, EDDHA are carcinogenic and toxic to human beings23. In order to avoid such unwanted risks, we have chosen greener chelators like citric acid for our study. In this study, we also formulated the combination of Fe and Zn nanocitrates and examined their effects on the plant along with individual metal citrates. The impact of single and combined Fe and Zn nanocitrates on groundnut plants growth and nutrient uptake through soil application and selected the best combinations that performed well for various characters considered. The entire plant was analyzed for potential variations in nutrient uptake and soil availability, including the leaching during the incubation period of iron and zinc after soil application of Fe and Zn nanocitrates.
Ferric nitrate hydrous (FN; ((Fe(NO3)3).9H2O)) purity of 98%, zinc nitrate hydrous (ZN; (Zn(NO3)2.6H2O)) purity of 98%, citric acid anhydrous (CA; (C6H8O7)) purity of 99% was purchased commercially from SRL, India. All the reagents were used as such without further purification.
Synthesis of combined iron (Fe) and zinc (Zn) based citrates
Ferric citrate (FC) (C6H5FeO7) and zinc citrate (ZC) ([C6H5O7]2Zn3) were prepared by a solid-state grinding technique as per our previous reports24.
\({Fe\left(N{O}_{3}\right)}_{3 }.{9H}_{2}\text{O}+ 3{C}_{6}{H}_{8}{O}_{7}+ {3Zn\left(N{O}_{3}\right)}_{2}.{6H}_{2}\text{O}\to {{{(C}_{6}{H}_{5}{O}_{7})}_{3}Fe.Zn}_{3}+{9HNO}_{3} \uparrow + {9H}_{2}O\) ------------- (1)
The combined citrates of iron and zinc were prepared by mixing ferric citrate (FC) and zinc citrate (ZC) of different weight ratios. The total weight was maintained as 10 gm. The resulting mixture was grounded for 45 minutes. The ground citrates were dried at 80°C in a hot air oven. The prepared citrates were further used in the studies without any further purification. The samples were coded as FZ (x:y) where x and y are the weights of FC and ZC respectively, used for the synthesis. These samples are referred to as prepared samples in the paper.
Synthesis of combined iron (Fe) and zinc (Zn) nanocitrates
The combined ferric and zinc nanocitrates were prepared through planetary ball-milling (XQM-1-A, Changsha Tianchuang Powder Technology Co. Ltd, China attached with a cooling unit to provide temperatures of 2–10°C) of FZ (x:y) for different hour duration. The different ball-milled samples were used as such for further studies. The ball-milled samples were coded as BFZ – X (X h = 2, 4, 6, 8, and 10 h).
Characterization of combined nanosystems
The as prepared and nanocitrates were characterized for their functional groups and particle size after completely drying. FTIR spectra were recorded using Thermo Nicolet (is50) spectrometer using diamond ATR crystal. Transmission Electron Micrographs (TEM) were taken on Tecnai T20, 200Kev, FEI. For Dynamic Light Scattering instrument (DLS) DLS characterization the samples were prepared by dispersing in pure isopropanol. The hydrodynamic size and zeta potential measurements were performed using a Malvern Zeta-Sizer NanoZS (Malvern Instruments, USA). The samples were equilibrated for 2 min at 25◦C before obtaining each measurement. For both TEM and DLS, the samples were dispersed at 0.01g/ ml in isopropanol. The X-ray fluorescence spectroscopy (XRF) was conducted using Panalytical Epsilon- 1 with omnion Fast Mode using Epsilon 3 software, directly measuring the weighed amount of sample for Fe and Zn content measurement.
Evaluation of combined Fe and Zn citrates
The combined Fe and Zn citrates were used to evaluate their effect on plant uptake, plant total dry matter, soil availability, and effect of soil leaching; the performance was compared with nano Fe and Zn citrates and commercial samples. A total 41 samples were applied to the red soil at a rate of 0.04 mmol of Fe/ kg of soil and 0.02 mmol of Zn/ kg of soil for citrate samples, and details were given in Table 1 and Table 2. The powder forms of samples were mixed in red soil uniformly. The entire experiment was conducted in a completely randomized design with three replicates.
| Unball milled samples | Ball milled samples | Commercial checks | |||
|---|---|---|---|---|---|
| FC (1:1) | BFC (1:1) -6 | BFZ (5:5) -2 | BFZ (2:8) -6 | BFZ (6:4) -10 | FeSO4 |
| ZC (1:3) | BZC (1:3) -6 | BFZ (5:5) -4 | BFZ (2:8) -8 | BFZ (4:6) -2 | ZnSO4 |
| FZ (8:2) | BFZ (8:2) -2 | BFZ (5:5) -6 | BFZ (2:8) -10 | BFZ (4:6) -4 | nano Fe- Geolife |
| FZ (6:4) | BFZ (8:2) -4 | BFZ (5:5) -8 | BFZ (6:4) -2 | BFZ (4:6) -6 | nano Zn- Geolife |
| FZ (5:5) | BFZ (8:2) -6 | BFZ (5:5) -10 | BFZ (6:4) -4 | BFZ (4:6) -8 | Fe-EDTA- Fe Gro |
| FZ (4:6) | BFZ (8:2) -8 | BFZ (2:8) -2 | BFZ (6:4) -6 | BFZ (4:6) -10 | Zn-EDTA- ZinGap |
| FZ (2:8) | BFZ (8:2) -10 | BFZ (2:8) -4 | BFZ (6:4) -8 | Control | |
| Treatment | Fe content/ kg of soil | Zn content/ kg of soil |
|---|---|---|
| FC (1:1) | 0.040 mmol | - |
| ZC (1:3) | - | 0.020 mmol |
| FZ (8:2) | 0.032 mmol | 0.004 mmol |
| FZ (6:4) | 0.024 mmol | 0.008 mmol |
| FZ (5:5) | 0.020 mmol | 0.010 mmol |
| FZ (4:6) | 0.016 mmol | 0.012 mmol |
| FZ (2:8) | 0.008 mmol | 0.020 mmol |
| FeSO4 | 0.016 mol | - |
| ZnSO4 | - | 0.015 mol |
| nano Fe | 0.025 mol | - |
| nano Zn | - | 0.025 mol |
| Fe-EDTA | 0.025 mol | - |
| Zn-EDTA | - | 0.025 mol |
Sedimentation studies
The sedimentation studies on the samples were analyzed as per the procedure reported in the earlier literature25. The samples were added to dry soil (400 mg/ kg). The soil extracts were prepared by weighing soil and added with water in the ratio of 1:5 and were allowed to shake for 5–6 h at 150 rpm. The soil suspensions were bath-sonicated (5 min) immediately before use in static sedimentation studies over a long period (30 days (d)). The sedimentation dynamics were measured using a Multi-Mode Microplate Reader spectrophotometer (BioTek, USA). The absorbance was measured at 600 nm. The sedimentation readings were taken at different weekly intervals. The patterns were inferred from the time course of normalized suspension absorbance at 600 nm (A/A0, where A0 was the initial absorbance at the start of the experiment). The absorbance of the filtered soil extract alone was also checked over time as a control experiment.
Incubation studies
About 500 g (oven dry weight) of soil (loamy sand alfisols having a neutral pH with more than 0.75% organic carbon content) were mixed separately with 2 g citrate and commercial samples, and filled in plastic containers (8 cm diameter × 15 cm length). The plastic containers were provided with a basal 1 cm glass wool pad over the drainage pores26. The measurements were made in triplicate. Initially, the red soil after nutrient treatments was allowed for incubation. The incubation was maintained for four days at field capacity with deionized water. A 25 mL of deionized water was added to the incubated soils to collect the leachates. The Fe and Zn content was determined using through Atomic Absorption Spectrophotometer (AAS). The Fe and Zn were also analyzed for soil before and after leaching.
DTPA extraction of Fe and Zn
An amount of 10 g of soil was collected and air-dried. The extraction of air-dried soil was done with 20 mL DTPA (Diethylenetriamine pentaacetate) for determining the available micronutrients in the soil26. The DTPA extracting solution was prepared by adding 13.3 ml of triethylamine (TEA), 1.967 g of DTPA, and 1.47 g calcium chloride dihydrate to one liter of water. The pH of the extract solution was adjusted to 7.3 and was shaken for 2 h on a rotary shaker. The supernatant collected from the soil sample was filtered through Whatman 42 filter paper and used for AAS analysis.
Evaluation of combined Fe and Zn citrates on plant growth of groundnut
Groundnut (Arachis hypogaea var. kadiri-6- registered variety) seeds were used in this study was obtained from seed unit section of ICAR-Indian Institute of Oilseeds Research, Hyderabad. The experiment on plants was conducted as per the institutional and national guidelines and legislation. Seeds were sown in small pots in controlled conditions of seed incubator on 1 kg red soil (2 mm sieved) maintained for 20 days old seedlings. The groundnut seeds were surface sterilized with sodium hypochlorite solution of 0.4% for 60 s and then rinsed immediately with distilled water to protect seedlings from any kind of diseases. About 15 seeds of groundnut were sown in pots and maintained in a seed incubator having a temperature of 27 ± 1 ⁰C for 20 days. No visual symptoms of deficiency or toxicity of Fe and Zn were observed. The experiment was conducted in triplicates. The seedlings were watered with an equal amount of deionized water based on the requirement in all treatments. On the 21st day, no. of germinated seedlings was counted, rinsed thoroughly with deionized water, and taken the shoot and root length of each seedling. For dry weight determination, the seedlings were dried at 60–70 ºC for 48 hours24.
Atomic absorption spectrophotometer (AAS) analysis
The dried plant samples were grounded in pestle-mortar for mineral analysis until the dry powder was obtained. After that, the sample (0.5 g) was digested using a 10 mL mixture of concentrated nitric acid and concentrated perchloric acid in the ratio of 9:1 on a hot plate having a temperature controller (JS Instruments, India) until clear and white fumes had resulted. After digestion, the digest filtrate was made up to 100 mL with deionized water. Appropriate dilutions were carried out to analyze Fe and Zn with AAS24.
The atomic absorption spectrometer (Perkin Elmer PinAAcle 900F, USA) was used to determine Fe and Zn with an air-acetylene flame. For Fe, the instrumental parameters employed were as follows: wavelength 248.3 nm; lamp current 12 mA; bandpass 0.2 nm; a hollow cathode lamp for the background correction. For Zn, the instrumental parameters were set: wavelength 213 nm; lamp current 12 mA; bandpass 0.2 nm; a hollow cathode lamp for the background correction. The calibration graph was obtained by feeding working standards of 0.5, 1.0, 2.0, and 3.0 mg/L for Fe and 0.2, 0.4, 0.6, and 0.8 mg/L, for Zn. Working standards for calibration were obtained by appropriate dilution of stock solution (1000 mg/L) of Fe and Zn (Perkin Elmer Pure AAS Standards) using deionized water. The WINLAB 32 AA Version 7.4.1 software was used for final quantification.
Statistical data analysis
The analytical determination was carried out from the average mean data of 45 plants in a set (control and treated). All experimental data were expressed as mean of replicates and analyzed using analysis of variance (ANOVA) using R version 4.0.4 software.
The contribution of each character to the total variation of all genotypes by the principal component analysis (PCA). The principal components (PCs) were evaluated using R version 4.0.4 software.
It is difficult to conclude the best performer for all the characters by considering the performance of treatment for each character separately. The best performers were identified by constructing a combined weighted index for all the studied characters. We have developed a weighted index approach based on the principal components in the present study. In this method, weights will be given for these physiological characters based on the principle that the variation in the linear combination of these characters should be maximum. The weighted index can be defined as
Solid state grinding followed by ball milling has been widely used for mechano-chemical synthesis27,28. In solid state grinding, metal citrates were obtained from metal nitrates and citric acid, alongside evolution of nitric acid fumes. It was demonstrated that mechanical energy of solid-state grinding can break the chemical bonds in solid materials and produce dangling bonds, which are very active for chemical reactions. The reaction has been confirmed by nitric acid fumes, color change and FTIR characterization.
Fe and Zn content in citrates
The Fe and Zn contents in developed series of combined and individual citrates were estimated using XRF. The Fe content ranged from 6 to 33%, whereas the Zn content ranged between 3 to 20%, which finds potential good sources of Fe and Zn (Table 3) as plant nutrients. The Fe and Zn content available within citrates are in the range of market available micronutrients and nano nutrients.
|
Treatment |
Fe content (%) |
Zn content (%) |
|---|---|---|
|
FC (1:1) |
33.16 |
0.007 |
|
ZC (1:3) |
0.012 |
18.28 |
|
FZ (8:2) |
34.13 |
3.27 |
|
FZ (6:4) |
20.25 |
11.11 |
|
FZ (5:5) |
21.79 |
11.31 |
|
FZ (4:6) |
16.47 |
15.42 |
|
FZ (2:8) |
6.36 |
20.28 |
FTIR, TEM, and DLS analyses of combined citrates
The ferric citrate is amorphous in nature as observed in XRD. The zinc citrate is crystalline in nature, but the peaks correspond to excess citric acid added during synthesis24. Similarly, for combined metal citrates amorphous or crystalline nature can be confirmed through XRD based on its composition ratio. The frequency assignment of IR spectra for the combined citrates collected after completely drying is shown in Supplementary Table 1, and spectra are shown in Fig. 1. The characteristic strong doublet peak between 1700 to 1400 cm− 1 in all the samples is attributed to carboxylate groups. The FTIR spectra confirmed the coordination of the metals Fe3+ and Zn2+ to the citrates and their ligand structures. Not many differences are observed in FTIR spectra of ball-milled citrates, which is proved in our early reports24. The lower shift in peak from 1750 cm− 1 in treatments FZ (6:4) and FZ (8:2) was observed. It could be due to the citric acid content present in the Zn metal citrate.
Fig. 2 visualizes the TEM image of combined citrates before ball milling and after ball milling. The TEM images of combined citrates indicate that the samples are aggregated and have a spherical morphology. Particle size decreased due to ball milling, and aggregation was observed in all images. Yuan et al.,29 reported similar results for aggregation in iron nanoparticles.
The hydrodynamic diameters of the combined citrates were ranging between 204.7 nm to 1455 nm based on ratios of Fe and Zn and ball milling duration; the zeta potentials were determined majorly to be negative except positive for two samples ranging between − 8.3 mV to 0.3 mV which were depicted in Supplementary Table 2. The samples with Zeta potential values between − 30 mV to + 30 mV exhibit aggregation30. It should be noted that hydrodynamic size and zeta potential will depend upon dispersion medium, pH and concentration etc31.
Sedimentation studies were performed to explain the stability of the nanoparticles in soil extracts. Higher sedimentation was observed immediately after 7 days of incubation which may be due to aggregation of particles. The sedimentation was again analyzed to reduce on day 15 and day 30. There was not much difference between sedimentation rates during 15th and 30th day for all the treatments. Although the sedimentation rates for 15th and 30th day remained the same, there were few exceptions for certain treatments. In BFZ (4:6)- 6, the sedimentation has been increased from 0.14 to 0.26. In few treatments, the sedimentation rate varied from 15 to 30 days in Fe-EDTA (0.22 to 0.12) and BFZ (4:6)- 10 (0.15 to 0.08). The change in sedimentation rates could be due to a change in the concentration of dispersed nanoparticles. This change in concentration may be due to rapid aggregation and disaggregation from soil extract during sampling after incubation32. The sedimentation was measured using normalized absorbance at different intervals as given in Fig. 3.
Changes in DTPA extractable Fe and Zn content during incubation
DTPA extractable Fe and Zn content in soil significantly varied with treatment. Maximum DTPA-Fe content was recorded before incubation 98.58 mg/kg in Fe-EDTA (commercial) whereas the highest Fe content in citrates is 65.81 mg/kg in BFZ (4:6)- 8 treatments (149.3 mg/kg after incubation). There was a reduction of DTPA-Fe content at the end of the incubation period followed by leaching in Fe- EDTA (35.23 mg/kg) (supplementary Table 3). In most of the citrates the Fe content was increased after incubation followed by leaching in all samples except few samples like FC (1:1) (reduced from 53.5 to 39.1 mg/kg), BFC (1:1)-6 (reduced from 46.5 to 20.1 mg/kg), and BFZ (4:6)- 2 (reduced from 62.7 to 60.6 mg/kg). As per the earlier reports, the gradual reduction of DTPA-Fe after incubation and leaching compared to initial days of incubation owing to the interaction of released Fe with soil components33,34.
The highest DTPA-Zn was recorded in nano Zn (210.6 mg/kg) followed by zinc sulfate (117.9 mg/kg) and BZC (1:3)- 6 (66.2 mg/kg) as given in supplementary Table 3. A drastic reduction or even negligible amounts in DTPA-Zn after incubation followed by leaching could be due to washing out during leaching or retention of Zn in soils35. The retention of zinc could be due to adsorption in soil particles because of fixation or aging and is the cause of reduction36. The availability of DTPA- Fe and Zn contents varied with the ratio of Fe and Zn in citrates, their particle size, mobility, etc. There was a decrease in DTPA-Fe content with an increase in DTPA-Zn irrespective of composition of the combined citrates (Supplementary Table 3). The maximum Zn content was coupled with the minimum Fe content and vice versa, irrespective of treatment. In combined citrates, Fe content decreased with the increase of DTPA-Zn content and vice versa owing to the formation of franklinite type of minerals37. Maximum Zn+ 2 solubility was furthermore related to the solubility of franklinite (ZnFe2O4), which was controlled by the availability of Fe+ 3 being limited by the pH-dependent solubility of Fe(III) hydroxides38.
Leaching effect
Leaching studies indicate the solubility and mobility of nutrients in soil. The leaching studies give an idea about nutrient availability at very different rooting depths. Soluble Fe and Zn contents of nitrates, sulfates, and commercial nanonutrients varied significantly with different samples. In citrate samples, very less Fe leaching of below 10 ppm was observed in the BFZ (8:2)- 8 and BFZ (4:6)- 8 treatments, and Zn leaching of 2 ppm was observed in the treatments like BFZ (8:2)- 2, nano Zn, BFZ (5:5)- 4, BFZ (8:2)- 4, BFZ (2:8)- 6, BFZ (6:4)- 2, and BFZ (2:8)- 8. Soluble Fe content was highest observed in samples like Fe-EDTA (855.3 mg/l), FZ (6:4) (382.6 mg/l), ferrous sulphate (134.9 mg/l) and nano Fe (159.5 mg/l); whereas in case of Zn, the highest soluble content was observed in Zn-EDTA (280.4 mg/l), ZC (1:3)- 0 (172.77 mg/l), zinc sulphate (141.63 mg/l) and nano Zn (164.41 mg/l) as shown in Supplementary Table 3. On average, the amount of soluble Fe content varied from 3.87 to 855.33 mg/l and soluble Zn content varied from 0.34 to 280.43 mg/l. The type of soil, organic matter, and easy water movement through the soil provide less time for Fe or Zn sorption resulting in a high amount of Fe and Zn leaching39. Sometimes, the percentage of nutrient leaching does not depend upon the nutrient application rate but on a specific soil's nutrient retention capacity. As per previous reports, any micronutrient's availability, sorption, leaching, and release depend upon soil properties40,41.
Evaluation of effect of combined Fe and Zn citrates on plant growth of groundnut
Groundnut seeds responded variably towards treatment with various Fe and Zn citrates along with commercial samples. Significant highest germination (80%) was observed in samples like FZ (8:2)- 0, BFC (1:1)- 6, BZC (1:3)- 6, BFZ (2:8)- 2, BFZ (4:6)- 2 and nano Zn and highest seedling vigor index in BZC (1:3)- 6 (3012.84) treatment. The seedling quality of all samples including the commercials and the untreated ones are presented in Supplementary Table 3. The germination was 40%, and the vigor index was 727.33 for untreated groundnut seedlings. However, although we have observed improvement in the germination and vigor index of almost all samples, few samples performed even less than the values of control experiments. Lin and Xing42, reported such inhibitory effects of nanoparticles on radish, grape, and ryegrass. The germination and vigor index improvement could be ascribed to higher precursor activity of plant growth enzymes due to Fe and Zn. Fe and zinc are some of the essential nutrients required for plant growth. It is an important component of various enzymes responsible for driving many metabolic reactions in all crops. Moreover, germination enhancement of ball milled samples may be due to increase in water uptake capacity of seed. This increase in water uptake capacity may be promoted by nanoparticles which create new pores on the seed coat43. Even though the prepared citrates are water soluble, with increase in ball milling duration, the solubility of the citrates has been reduced. The reduction in water solubility of ball milled citrates will keep it in nanosized and its entry into plants as nanoparticles.
The effect of Fe and Zn citrate nanoparticles and as prepared citrate particles on root and shoot length of groundnut is presented in the form of root to shoot ratio in Supplementary Table 3. Data of the table depict that root length of citrate-treated groundnut is drastically less in almost all samples, with the least root to shoot ratio observed in BFZ (8:2)- 4 (0.34), whereas few samples exhibited a higher root to shoot ratio as in BFZ (4:6)- 4, BFZ (6:4)- 10, BFCZ (1:1:1)- 10, FeSO4 and BFZ (5:5)- 2. The highest root to shoot ratio in BFZ (5:5)- 2 nanoparticle treated plants was only 1.14, but more than that of plants in the control experiment. (0.76). The root to shoot ratio decreased with increasing ball milling duration up to a certain level and decreases later on. The root to shoot ratio decreased with ball milling duration time between 4–6 hours and then it increased further up till 10 hours. This could be due to aggregation and moisture sensitivity in 10 h ball milled samples. This trend was observed in hydrodynamic diameter (Supplementary Table 3).
An increase in fresh and dry weight of treated plants was observed in all samples compared to control experiments except for very few samples (Supplementary Table 3). The samples that exhibited the highest and lowest fresh weights were FZ (8:2) (12.52 g) and BFZ (8:2)- 4 (2.42 g), followed by BFZ (6:4)- 4 (1.88 g) and BFZ (8:2)- 4 (0.3 g). The control samples exhibited fresh and dry weights were 3.53 g and 0.34 g respectively.
Fe and Zn Uptake by plants:
The total Fe and Zn content in the groundnut plants significantly increased due to citrate presence, the uptake is even more pronounced in nanocitrates which is more than their respective as-prepared counterparts (Supplementary Table 3). More Fe was taken up by plants than Zn, probably because Fe is required from the initial germination stage of the plants, and at the same time, the Fe content is more than Zn content in the samples. Fe uptake was higher in the BFZ (2:8)- 8 sample (0.48 mg of Fe/ pot) and Zn uptake is higher in nano zinc (commercial) (88.47 µg of Zn/ pot) followed by ZC (1:3)-0 (82.32 µg of Zn/ pot) samples. The control values were (0.02 mg of Fe/ pot and 2.05 µg of Zn/ pot).
The Zn accumulation is higher in nano zinc treatment, which is due to its lesser leaching effect. In ZC (1:3) treatment, the high Zn accumulation in the plant is due to its high percentage of Zn content and also less affected due to leaching. The Fe content is available in the soil even after leaching. The total Fe and Zn content observations revealed that the Fe and Zn contents in shoots were less in commercial samples (ferrous sulfate, zinc sulfate, nano zinc, nano iron, chelated Fe, and chelated Zn) compared to metal citrates, indicating that citrate NPs and their as prepared counterparts promoted the groundnut plants. The growth promotion in the case of nanocitrates could be due to the size of the particle, mobilization, and availability and less fixation (Fig. 4). Instead of analyzing the total Fe and Zn content in a 20-day plant, further study should focus on the Fe and Zn contents at different phases of the plant till crop duration, which can provide more information on complete usage.
Validation
The data for all 14 characters are represented in Supplementary Table 1 and were studied for all 53 samples, which were validated by principal component analysis (PCA) and ranking index.
Principal Component Analysis
The data obtained for 53 treatments including different mole ratios of ferric nitrate, citric acid and zinc nitrate was formulated and coded as FCZ (1:1:1) and FCZ (1:2:1) were subjected to PCA. These samples were ball milled at different durations as like other ratios, were subjected to PCA to unravel the underlying relationship among various plant and soil nutrient parameters. The weight ratio Fe and Zn is almost unity and thus similar to FZ(5:5). Eigenvalue obtained from PCA for plant quality characteristics, soil nutrient, and plant nutrient content is presented (Fig. 5A). The PC1, PC2, PC3 and PC4 contributed 31.2%, 21.8%, 10.5% and 10.1% of total variation, respectively (Fig. 5A). The important variables in principal component analysis were total fresh weight (gm), leachate- Zn (mg/kg), leachate- Fe (mg/kg) and plant Fe content (mg/ pot) for PC1, PC2, PC3 and PC4 respectively. The cluster analysis was performed to divide data into clusters (Fig. 5B) and represent the board groups and performance. Cluster- I and cluster- III contain the most of samples. Cluster- II contains samples that are equivalent to control treatments. Cluster I contains the best zinc treatments and cluster III the best iron treatments.
Ranking index
The samples for the studied characters were ranked through the ranking index and are represented in Fig. 6. The highest performer in all characters was FZ (8:2), followed by BFZ (4:6)- 2. There were some negatively ranked (11 samples) among 53 samples. Negative ranking means lower performance compared to untreated and commercial checks. The studied characters like germination (%), vigour index, fresh weight and dry weights played major role in ranking index.
We have successfully synthesized a series of combined citrates that is functionally valuable for plant use efficiency through soil application. Our results suggest that combined citrates have the potential to improve seed quality and plant uptake of Fe and Zn nutrients similar to their respective individual nutrients and other commercial fertilizers. These nanocitrates, along with their as prepared counterparts, were applied on groundnut and showed maximum nutrient use efficiency and higher dry matter. It was noticed that citrate increased Fe2+ and Zn2+ nutrient uptake efficiency more than commercial fertilizer within a few days. The best treatments were identified from this work through statistical ranking, physical observation and from important characters. Best ball milled treatments, BFC (1:1)- 6; BZC (1:3)- 6; BFZ (4:6)- 8; BFZ (5:5)- 2; BFZ (5:5)- 6; BFZ (8:2)- 4 was being considerate for our future work based on its performance in important parameters. This work concluded that Fe and Zn citrates individually and combined with as prepared counterparts and the respective nanosize has excellent advantages as a fertilizer, including low cost, nutrient- uptake, and negligible land contamination. Overall, the nanocitrates have found significant results compared to all other treatments.
Data availability: Data sets analyzed during the current studies will be available from the corresponding author on reasonable request.
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No competing interests reported.