Abstract
Background
Sustainable micronutrient management is vital for early crop establishment in legumes, as conventional molybdenum fertilizers often exhibit low bioavailability during early growth stages. In legumes, molybdenum plays an important role in nitrogen fixation and root nodule functioning through nitrogenase and nitrate reductase enzymes. Therefore, phytochemical-mediated molybdenum-based nanoparticles were synthesized and evaluated as a seed treatment for improving seedling vigour and early establishment of greengram (Vigna radiata L.) during 2024–2025 at the Department of Agronomy, University of Agricultural Sciences, GKVK, Bengaluru, India.
Results
The research examined how the phytochemical composition involved in the green synthesis of molybdenum-based nanoparticles influences seedling vigour responses in greengram (Vigna radiata L.). Molybdenum-based nanoparticles were synthesized using aqueous extracts of six plant species (Azadirachta indica, Centella asiatica, Citrus limetta, Psidium guajava, Simarouba glauca and Vitex negundo) with ammonium heptamolybdate as the precursor. Differences in phenolic and flavonoid content among extracts influenced nanoparticle formation, which was characterized using UV-Visible spectroscopy, particle size analysis, SEM, EDX and FTIR. A marked reduction in phenolic and flavonoid contents after synthesis confirmed the direct involvement of phytochemicals in nanoparticle reduction and stabilization. Among the tested extracts, Vitex negundo produced comparatively smaller and more uniform nanoparticles, with a Z-average particle size of 17.13 nm and a dominant size distribution peak accounting for 79.5% intensity, and was therefore selected for biological evaluation. Seed priming with green synthesized molybdenum-based nanoparticles resulted in clear improvements in seedling growth parameters without affecting germination percentage. The highest shoot length (18.84 cm), root length (16.49 cm) and seedling vigour index (3533) were recorded when seeds were primed at 200 ppm for 30 min, representing a substantial increase over hydro-primed control seedlings. Lower concentrations produced moderate improvements, whereas concentrations above 200 ppm caused a decline in shoot and root growth, indicating a narrow optimal dose range and phytotoxicity at higher concentrations.
Conclusions
These findings demonstrate that plant-based phytochemicals strongly influence the green synthesis of molybdenum-based nanoparticles and its performance and that optimized molybdenum-based nanoparticles priming can significantly enhance early seedling vigour, offering a sustainable strategy for improving early crop establishment in legume-based agroecosystems.
Keywords: Green synthesis, Molybdenum-based nanoparticles, Phytochemicals, Particle characterization, Seed priming, Seedling vigour
Background
The soil degradation, nutrient depletion and rising food production demand have made sustainable agriculture a necessity on a global scale. Micronutrients determine the health and yield of plants to a large extent and molybdenum (Mo) is one of the essential micronutrients since it serves as a constituent of the molybdenum cofactor (MoCo), which is essential for the activation and takes part in a number of enzymatic processes like reduction of nitrate, fixation of nitrogen, sulfur metabolism and effective root nodule development in legumes [1]. Molybdenum deficiency leads to stunted growth, poor nodulation and low yields, especially in legumes. However, traditional molybdenum fertilizers often have very limited bioavailability and poor solubility, leading to inefficient nutrient use and environmental loss.
Nanotechnology is a powerful strategic method that has been at the forefront of development when it comes to conquering the conventional nutrient management barriers i.e., better nutrient use efficiency, better uptake process and even higher plant tolerances to environmental stresses. Due to their small size, nanoparticles of molybdenum (MoNPs) are known to be much more efficient in terms of bioavailability and plant uptake compared to the traditional larger molybdenum forms. Among the various methods of synthesis available, the green synthesis route using plant extracts has become very popular due to its environmental friendliness, cheapness and ease of operation. The phytochemicals such as phenolics, flavonoids, terpenoids and alkaloids, which are naturally present in the plant extracts, play a crucial role in the eco-friendly manufacture of nanoparticles by acting as reducing and stabilizing agents without the use of toxic reagents [2, 3].
Different plant species vary in phytochemical composition and concentration and these variations are very important factors that influence the morphology, stability and functional properties of the nanoparticles synthesized [4–7]. Various metal nanoparticles via green synthesis have been researched in great detail but there are hardly any comparative reports on the synthesis of molybdenum-based nanoparticles from different plant sources, especially concerning the effect of phytochemical diversity on the characteristics of the nanoparticles. Moreover, there has not been much research done to relate the detailed characterization of nanoparticles to their biological performance in plants. It is very important to comprehensively understand these interrelationships in order to rationally develop efficient, plant-mediated nanomaterials with potential applications in agriculture.
The purpose of this research was to find out whether six plant species, namely Azadirachta indica (neem), Centella asiatica (Indian pennywort), Citrus limetta (sweet lemon), Psidium guajava (guava), Simarouba glauca (paradise tree) and Vitex negundo (chaste tree) could be used for the green synthesis of molybdenum-based nanoparticles. These particular plant species were picked because of their diverse and rich phytochemical profiles which are known to facilitate the formation and stabilization of nanoparticles [8–12]. The nanoparticles produced were characterized step by step in order to measure their structural, morphological and compositional aspects. A comparative analysis was performed on the six plant-mediated nanoparticles to find out which extract was the best for generating stable and uniform molybdenum-based nanoparticles. The nano formulation obtained from Vitex negundo was further subjected to a germination assay with greengram to study its influence on seed germination and early seedling development. This research sheds light on the role of phytochemical diversity in determining the synthesis and properties of molybdenum-based nanoparticles and at the same time indicates the possible future use of this technique in sustainable agriculture.
Materials and methods
Site of experiment
The experimental study was conducted at the University of Agricultural Sciences (UAS), Bangalore, India, under controlled laboratory and experimental conditions.
Plant materials collection
Healthy and fully matured leaves of Azadirachta indica, Centella asiatica, Psidium guajava, Simarouba glauca, Vitex negundo and pith of Citrus limetta were utilized in this investigation. The samples were collected from the cultivated species at University of Agricultural Sciences, Bengaluru, India. Plant sample collection and experimental work were conducted in adherence to institutional guidelines. The plant materials were identified based on standard institutional records at UAS, Bengaluru. No material was collected from the wild therefore no special permits were needed for the sample collection. The study was conducted in accordance with the local legislation.
Reagents
The primary precursor used for molybdenum-based nanoparticles synthesis was ammonium heptamolybdate [(NH₄)₆Mo₇O₂₄·4 H₂O] (assay: 99.0-102.0%). The estimation of total phenolic content (TPC) and total flavonoid content (TFC) utilized analytical grade reagents, which included Folin-Ciocalteu reagent (FCR), gallic acid, quercetin, aluminum chloride (AlCl3), sodium carbonate (Na2CO3) and methanol.
Preparation of extract solution
Fresh leaves (Azadirachta indica, Centella asiatica, Psidium guajava, Simarouba glauca and Vitex negundo) and pith (Citrus limetta) of the selected plant species were employed as biological precursors for the green synthesis of molybdenum-based nanoparticles. The collected plant materials were properly rinsed with tap water, followed by distilled water, to remove dust particles and surface impurities. After being cleaned, the samples were dried in the shade at ambient temperature for five to seven days and subsequently oven-dried at 60 °C to eliminate residual moisture. The dried materials were ground very fine with an electric grinder and stored in airtight containers until further use. For extract preparation, 200 mL of distilled water was mixed in 10 g of the powdered plant material and stirred on magnetic hot plate at 650 rpm for 30 min at boiling temperature. The resulting mixture was filtered through Whatman No. 1 filter paper to obtain a clear plant extract, which was then used for nanoparticle synthesis.
Ammonium heptamolybdate [(NH₄)₆Mo₇O₂₄·4 H₂O] solution preparation
A precisely weighed quantity of 1 g ammonium heptamolybdate [(NH₄)₆Mo₇O₂₄·4 H₂O] was dissolved in 100 mL of distilled water and stirred continuously for 15 min to ensure complete dissolution and formation of a homogenous solution.
Synthesis of green molybdenum-based nanoparticles
An aliquot of 100 mL of the filtered plant extract was combined with the ammonium heptamolybdate solution in a 1:2 proportion (extract to salt solution). The resulting mixture was heated to boiling under constant stirring at 650 rpm on a magnetic hot plate for 2 h (Fig. 1).
Fig. 1.
Schematic representation of molybdenum-based nanoparticles synthesis from various plant biomass
Characterization of synthesized molybdenum-based nanoparticles
To ascertain the physicochemical characteristics of the produced molybdenum-based nanoparticles, a variety of analytical methods were used. The pH of the plant extracts and synthesized molybdenum-based nanoparticles solutions was measured using a digital pH meter (FiveEasy Plus, Mettler Toledo). UV-Visible spectroscopy (Genesys 150, Thermo Fisher Scientific, USA) was performed in 2 nm interval and the wavelength range of 200–800 nm to identify the surface plasmon resonance (SPR) peaks characteristic of molybdenum-based nanoparticles. Particle size distribution was measured at 25 ± 1 °C using a Zetasizer Nano ZS-90 (Malvern Instruments, UK), employing water as the dispersant (refractive index 1.330; viscosity 0.8872 cP). Morphological analysis was performed with the help of Scanning Electron Microscopy (SEM; EVO 18, Standort Oberkochen, Germany) which enabled the visualization of surface structure and particle aggregation, while the elemental composition was verified through Energy-Dispersive X-ray spectroscopy (EDX; EVO 10, Carl Zeiss Microscopy, Germany). The total phenolic content (TPC) and total flavonoid content (TFC) of both plant extracts and nanoparticles were determined using the Folin-Ciocalteu and aluminum chloride colorimetric methods, respectively [13]. Gallic acid and quercetin were the standard references and the results were expressed as µg gallic acid equivalents (GAE) and µg quercetin equivalents (QE) per gram of sample. Nanoparticle synthesis and stabilization were supported by these stabilis which gave the researchers access to the phytochemical components that played the major role in those processes.
Determination of molybdenum-based nanoparticles concentration
The plant samples were first shade dried and then dried in an oven at a temperature of 65–70 °C until constant weight was achieved to remove residual moisture before grinding and mineral analysis, and then milled into a fine powder. A portion of about 1.0 g of the powdered substance made from plant samples was subjected to digestion in a mixture of concentrated nitric acid (HNO₃) and perchloric acid (HClO₄) in a 9:4 proportion in a digestion flask. The mixture was eventually heated gently until the solution was clear indicating complete digestion. Subsequent to cooling, the digest was filtered and diluted to a predetermined volume with deionized water. The Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES; PerkinElmer Optima 8000, USA) was used to determine the molybdenum concentration in the samples. Molybdenum determination was carried out at its characteristic emission wavelength of 204.597 nm, chosen to reduce spectral interference and enhance analytical accuracy. A spectral window of 204.506–204.688 nm was employed and automatic background correction was applied during the measurements.
Seed germination study
The assessment of greengram seed germination and early seedling growth was done at Department of Agronomy, University of Agricultural Sciences, Gandhi Krishi Vigyan Kendra, Bengaluru, to determine the influence of synthesized molybdenum-based nanoparticles from Vitex negundo using the paper towel method according to the International Seed Testing Association (ISTA) standards and previous studies [14, 15]. The experiment utilized greengram (Vigna radiata L.) variety KKM-3 (Karnataka Kathalagere Moong-3), a short-duration (60–65 days) improved cultivar released from the Agricultural Research Station (ARS), Kathalagere, Karnataka, in 2009. The variety has a yield potential of 7.5–8 q ha⁻¹ under rainfed conditions and exhibits moderate tolerance to Yellow Mosaic Virus, powdery mildew, and pod borer. The seeds were procured from the NSP (National Seed Project) Unit, University of Agricultural Sciences (UAS), Bangalore, India.Calibration was done with standard molybdenum solutions and the results were given in parts per million (ppm). The synthesized molybdenum-based nanoparticle suspension served as the stock solution for the biological assays. Working concentrations of 50, 100, 150, 200, 250, 300 and 350 ppm were prepared by serial dilution of the nanoparticle stock using the dilution equation N₁V₁ = N₂V₂. The reported ppm values represent the concentration of the synthesized molybdenum-based nanoparticle suspension. A completely randomized design (CRD) was deployed with two priming times of 15 and 30 min with three replications for each priming time. In each replication, ten seeds were positioned between two brown corrugated paper towels which were lightly moistened to prevent excess water while providing sufficient hydration. A transparent plastic was used for the first sheet, seeds were distributed evenly and then covered with the second moistened sheet. The towels were cylindrical, wrapped with butter paper, kept in place with rubber bands and put vertically in a seed incubator where the temperature ranged from 20 to 30 °C and humidity, air circulation were controlled. All the viable seeds were sprouted and seed germination was noted daily until then. After seven days, shoot length, root length, germination percentage and Seed Vigour Index (SVI) were recorded. The calculation of germination percentage was made by considering the total number of seeds germinated and the seed vigour index was calculated taking a standard formula [15].
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Statistical analysis
The data that was gathered through the experiment was analyzed using the analysis of variance (ANOVA) technique under a Completely Randomized Design (CRD). For this purpose, Duncan’s Multiple Range Test (DMRT) at p ≤ 0.01 was employed for mean separation to differentiate significant differences among the treatments. All the statistical calculations were done with the help of R software (version 4.5.1).
Results
Visual observation and preliminary confirmation of nanoparticle formation
The synthesis processes were studied in terms of color changes and pH, which is summarized in Table 1 and illustrated in Fig. 2. When the colorless ammonium heptamolybdate solution was mixed with the plant extract, a pH shift and gradual coloring were observed for up to 2 h, which was an indication of the beginning of the reactions driven by plant chemicals that resulted in the formation of nanoparticles [9, 15, 16]; however, there was no significant discoloration after 24 h which implied that the reaction had completed.
Table 1.
Changes in color and pH during the synthesis of molybdenum-based nanoparticles
| Plant sample | Color | pH | ||
|---|---|---|---|---|
| Initial | Final | Initial | Final | |
| Azadirachta indica | Light brown | Reddish brown | 7.41 | 6.04 |
| Centella asiatica | Brown | Dark brown | 6.72 | 5.98 |
| Citrus limetta | Pale yellow | Dark teal | 7.01 | 5.97 |
| Psidium guajava | Brown | Dark brown | 6.71 | 5.94 |
| Simarouba glauca | Light brown | Dark brown | 7.02 | 5.89 |
| Vitex negundo | Dark brown | Dark teal | 7.39 | 6.03 |
Fig. 2.
Synthesis of molybdenum-based nanoparticle using various plant biomass. a Plant extract. b Ammonium heptamolybdate aqueous solution. c Synthesized molybdenum-based nanoparticles
UV-Vis spectral analysis
The UV-Vis spectral analysis was performed on plant extracts, ammonium heptamolybdate and their corresponding synthesized molybdenum-based nanoparticles suspensions (Fig. 3). Characteristic absorption peaks of the plant extracts of Azadirachta indica (224 nm) (Fig. 3a), Centella asiatica (214 nm) (Fig. 3b), Citrus limetta (210 nm) (Fig. 3c), Psidium guajava (242 nm) (Fig. 3d), Simarouba glauca (230 nm) (Fig. 3e) and Vitex negundo (228 nm) (Fig. 3f) were found between 200 and 300 nm that were the consequence of phenolics and flavonoids being present. A solution of ammonium heptamolybdate presented a classic absorption maximum at 230 nm, which acted as the precursor reference. With the reduction and stabilization by the particular plant extracts, the produced molybdenum-based nanoparticles samples displayed absorption peaks clearly at Azadirachta indica (228 nm) (Fig. 3a), Centella asiatica (230 nm) (Fig. 3b), Citrus limetta (230 nm) (Fig. 3c), Psidium guajava (248 nm) (Fig. 3d), Simarouba glauca (242 nm) (Fig. 3e) and Vitex negundo (235 nm) (Fig. 3f).
Fig. 3.
Comparative UV-Vis spectra of plant extract, ammonium heptamolybdate and synthesized molybdenum-based nanoparticles obtained using various plant biomass. a UV-Vis spectra of Azadirachta indica. b UV-Vis spectra of Centella asiatica. c UV-Vis spectra of Citrus limetta. d UV-Vis spectra of Psidium guajava. e UV-Vis spectra of Simarouba glauca. f UV-Vis spectra of Vitex negundo
Particle size analysis (PSA)
The particle size distribution of molybdenum-based nanoparticles produced through synthesis was examined utilizing a Particle Size Analyzer, and the outcomes are shown in Table 2; Fig. 4. The investigation disclosed that all the samples derived from plants displayed multi-modal size distributions, which portrayed the existence of nanoparticles with different levels of aggregation. The Z-average particle sizes varied from about 13.02 to 49.92 nm, with Simarouba glauca (13.02 nm) exhibiting smallest Z-average particle size followed by Azadirachta indica (15.03 nm), Vitex negundo (17.13 nm), Centella asiatica (26.57 nm), Psidium guajava (26.76 nm) and Citrus limetta (49.92 nm). The DLS intensity distribution further demonstrated differences in nanoparticle homogeneity among the plant extracts. The proportion of the primary nanoparticle population (Peak 1 intensity) was highest in Citrus limetta (93.6%), followed by Centella asiatica (91.1%), Vitex negundo (79.5%), Azadirachta indica (66.5%) and Simarouba glauca (66.5%), while the lowest proportion was observed in Psidium guajava (49.7%). However, the overall particle distribution also depended on the number, size, and intensity of secondary and tertiary peaks, which represent aggregated particle populations. The DLS intensity distribution showed that Vitex negundo possessed a dominant nanoparticle population at 19.07 ± 11.71 nm accounting for 79.5% of the total scattering intensity, with only one secondary peak (514.2 ± 334.9 nm; 20.5% intensity), indicating a relatively homogeneous particle-size distribution and lower aggregation. In contrast, Simarouba glauca exhibited three distinct particle populations, with only 66.5% of the intensity corresponding to the primary nanoparticle peak, while the remaining intensity was distributed among larger aggregates (1610 ± 586.6 nm; 25.6% intensity) and an additional minor population (2.933 ± 0.6125 nm; 7.9% intensity), reflecting greater heterogeneity. Similarly, although Centella asiatica (91.1%) and Citrus limetta (93.6%) exhibited higher primary peak intensities, both contained very large secondary particle populations (4822 ± 712.9 nm and 4419 ± 913.3 nm, respectively), indicating the presence of substantial aggregates. Likewise, Psidium guajava showed the greatest heterogeneity with three particle populations and only 49.7% of the scattering intensity corresponding to the principal nanoparticle fraction, while Azadirachta indica also displayed three distinct populations, reflecting considerable aggregation.
Table 2.
Particle size of molybdenum-based nanoparticles in selected plant samples
| Plant sample | Z-Average (nm) | Peak 1 (nm, % Intensity) | Peak 2 (nm, % Intensity) | Peak 3 (nm, % Intensity) |
|---|---|---|---|---|
| Azadirachta indica | 15.03 | 13.45 ± 4.208 (66.5) | 1857 ± 678.5 (25.6) | 3.273 ± 0.6067 (7.9) |
| Centella asiatica | 26.57 | 24.95 ± 7.896 (91.1) | 4822 ± 712.9 (8.9) | - |
| Citrus limetta | 49.92 | 77.90 ± 55.35 (93.6) | 4419 ± 913.3 (6.4) | - |
| Psidium guajava | 26.76 | 99.09 ± 62.85 (49.7) | 14.42 ± 6.813 (39.6) | 3795 ± 1156 (10.7) |
| Simarouba glauca | 13.02 | 11.66 ± 3.691 (66.5) | 1610 ± 586.6 (25.6) | 2.933 ± 0.6125 (7.9) |
| Vitex negundo | 17.13 | 19.07 ± 11.71 (79.5) | 514.2 ± 334.9 (20.5) | - |
Fig. 4.
Particle size analysis (PSA) of synthesized molybdenum-based nanoparticles from various plant biomass. a PSA of Azadirachta indica. b PSA of Centella asiatica. c PSA of Citrus limetta. d PSA of Psidium guajava. e PSA of Simarouba glauca. f PSA of Vitex negundo
Therefore, Vitex negundo was selected for further physicochemical characterization because it provided the best overall balance between nanoscale particle size and particle-size homogeneity. Although it did not exhibit the smallest Z-average particle size or the highest primary peak intensity, its DLS profile consisted of a single dominant nanoparticle population accompanied by only one comparatively smaller aggregate population, without the additional tertiary peaks observed in several other extracts. In contrast, the remaining plant extracts either exhibited greater polydispersity with multiple particle populations or contained large micron-sized aggregates despite having higher primary peak intensities. Consequently, Vitex negundo demonstrated the lowest overall aggregation and the most uniform nanoparticle dispersion, making it the most suitable candidate for subsequent physicochemical characterization.
Total Phenolic Content (TPC) and Total Flavonoid Content (TFC)
The total phenolic content (TPC) determined for the six plant extracts showed large variations, with the highest concentrations of phenolic being recorded for Simarouba glauca (1809.29 µg GAE g− 1) and Citrus limetta (152.26 µg GAE g− 1) giving the lowest values relatively. After the nanoparticle synthesis, the TPC values decreased considerably, with the highest reduction shown in Centella asiatica (61.87%), followed by Vitex negundo (57.45%), suggesting that more of their phenolic components were involved in the molybdenum ion reduction. On the other hand, Citrus limetta (17.22%) showed relatively lower reduction percentages (Table 3).
Table 3.
Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) of plant extracts and molybdenum-based nanoparticles
| Plant sample | Total Phenolic Content (µg GAE g− 1) |
Total Flavonoid Content (µg QE g− 1) |
||||
|---|---|---|---|---|---|---|
| Plant extract | Nano-Mo (free TPC) | Reduction (%) | Plant extract | Nano-Mo (free TFC) | Reduction (%) | |
| Azadirachta indica | 450.43 | 317.97 | 29.41 | 299.76 | 162.71 | 45.72 |
| Centella asiatica | 385.12 | 146.85 | 61.87 | 274.00 | 135.54 | 50.53 |
| Citrus limetta | 152.26 | 126.04 | 17.22 | 29.19 | 10.55 | 63.75 |
| Psidium guajava | 1248.80 | 815.64 | 34.69 | 322.95 | 154.17 | 52.26 |
| Simarouba glauca | 1809.29 | 1101.90 | 39.10 | 177.61 | 113.22 | 36.25 |
| Vitex negundo | 317.02 | 134.89 | 57.45 | 156.46 | 133.20 | 14.87 |
| F- test | * | * | * | * | * | * |
| S.Em± | 0.54 | 0.61 | 0.20 | 0.58 | 0.64 | 1.14 |
| CD @ 1% | 2.35 | 2.63 | 0.87 | 2.49 | 2.76 | 4.30 |
Values represent the mean of three independent replicates (n = 3). S.Em± denotes standard error of the mean. CD denotes critical difference at 1% level of significance. Plant extract values stand for the initial Total Phenolic Content (TPC) and Total Flavonoid Content (TFC). Nano-Mo values denote the free phenolics and flavonoids that are left after the synthesis. Reduction (%) reflects the amount of phenolic and flavonoid compounds used as reducing agents and stabilizing agents in the creation of the Nano-Mo organic complex
* denotes significance at p ≤ 0.01
The total flavonoid content (TFC) of the plant extracts showed significant variation, with the lowest being 29.19 µg QE g− 1 in Citrus limetta and the highest being 322.95 µg QE g− 1 in Psidium guajava (Table 3). The highest flavonoid content was found in Psidium guajava (322.95 µg QE g− 1), while Citrus limetta (29.19 µg QE g− 1) had comparatively the lower levels. After the molybdenum-based nanoparticles synthesis, TFC values of all species decreased, with Citrus limetta (63.75%) showing the highest reduction. In contrast, Vitex negundo (14.87%) presented the minimal reduction.
Concentration of molybdenum in synthesized molybdenum-based nanoparticles
The molybdenum concentration in the molybdenum-based nanoparticles samples synthesized from six various plant extracts was measured by ICP-OES. The values obtained showed a range of molybdenum content in the plant-based nanomaterials, reflecting the differences in the efficiency of molybdenum incorporation during the nanoparticle synthesis (Table 4).
Table 4.
Concentration of molybdenum in synthesized nanoparticles
| Plant sample | Molybdenum (ppm) in synthesized molybdenum-based nanoparticles extract |
|---|---|
| Azadirachta indica | 20,380 ± 34.06b |
| Centella asiatica | 9585 ± 20.00f |
| Citrus limetta | 22,665 ± 50.00a |
| Psidium guajava | 15,465 ± 21.50c |
| Simarouba glauca | 13,455 ± 24.08e |
| Vitex negundo | 14,625 ± 20.00d |
Values are presented as mean ± standard error (SE) of five independent replicates (n = 5). Means followed by different superscript letters are significantly different according to one-way ANOVA followed by DMRT test at P < 0.01
Scanning electron microscopy (SEM)
The SEM micrographs of molybdenum-based nanoparticles synthesized using Vitex negundo extract revealed predominantly irregular shaped particles along with average sizes in the nanometer range (45.20 nm). The particles appeared well-dispersed, indicating the presence of phytochemicals acting as capping and stabilizing agents (Fig. 5a).
Fig. 5.
a SEM micrograph of molybdenum-based nanoparticles synthesized using Vitex negundo extract. b EDX of synthesized molybdenum-based nanoparticles from Vitex negundo
Energy Dispersive X-ray Analysis (EDX)
The Energy Dispersive X-ray (EDX) analysis confirmed the presence of molybdenum in the synthesized nanoparticles (Fig. 5b). The peaks corresponding to carbon (C), oxygen (O) and molybdenum (Mo), validating the successful synthesis of molybdenum-based nanoparticles. On weight basis, molybdenum, carbon and oxygen accounting for 25.27%, 43.58% and 31.14%, respectively.
Fourier Transform Infrared Spectroscopy (FTIR)
FTIR spectra of Vitex negundo extract and the synthesized molybdenum-based nanoparticles exhibited distinct features indicating functional groups and interactions presented in Fig. 6. The extract displayed characteristic peaks corresponding to O–H stretching vibrations around 3298.28 cm⁻¹, C = O amide I band stretching 1637.56 cm⁻¹ and Mo–O stretching near 437.84 cm⁻¹, confirming the presence of alcohols, phenolics, aromatics and metal oxides. In contrast, the molybdenum-based nanoparticles synthesized using Vitex negundo revealed a modified spectral pattern: peaks corresponding to O–H and C = O groups were slightly shifted, indicating their participation in the reduction and stabilization of molybdenum. Additionally, the pronounced Mo = O and Mo–O–Mo vibrations in the 950 –800 cm⁻¹ region confirmed the successful formation of molybdenum-based nanoparticles [17–19].
Fig. 6.
a FTIR spectra of Vitex negundo extract, b FTIR spectra of molybdenum-based nanoparticles from Vitex negundo
Effect of synthesized nano-mo on seed germination and seed vigour index of greengram
The germination percentage of greengram seeds exhibited numerical variation across treatments, ranging from 90 to 100 per cent (Table 5). However, the deviation among treatments was observed to be statistically non-significant. Shoot and root length of greengram seedlings showed significant difference across the treatments. The highest shoot length (18.84 cm) and root length (16.49 cm) were revealed in seed primed with synthesized molybdenum-based nanoparticles @ 200 ppm for 30 min, followed by seed primed with molybdenum-based nanoparticles @ 200 ppm for 15 min and seed priming with molybdenum-based nanoparticles @ 150 ppm for 30 min, which recorded shoot lengths (16.95 cm and 16.57 cm, respectively) and root lengths (16.05 cm and 15.78 cm, respectively). On the other hand, seed priming with distilled water for 15 min gave a lower shoot length of 10.89 cm and root length of 11.14 cm. The highly significant seed vigour index (3532.67) was recorded. in seeds that were primed with synthesized molybdenum-based nanoparticles @ 200 ppm for 30 min, which was statistically equivalent to seed primed with molybdenum-based nanoparticles @ 200 ppm for 15 min and seed primed with molybdenum-based nanoparticles @ 150 ppm for 30 min that recorded 3299.67 and 3234.67 respectively. In contrast, the very low seed vigour index of 1983 was recorded in seed priming with distilled water for 15 min (Fig. 7).
Table 5.
Effect of synthesized molybdenum-based nanoparticles on germination percentage, shoot and root length and seed vigour index
| Treatments | Germination % |
Shoot length (cm) | Root length (cm) | Seedling Vigour Index (SVI) |
|---|---|---|---|---|
| T1: Seed primed with synthesized molybdenum-based nanoparticles @ 50 ppm for 15 min | 93.33 ± 3.33a | 13.86 ± 0.32def | 14.14 ± 0.49de | 2609.63 ± 63.13fgh |
| T2: Seed primed with synthesized molybdenum-based nanoparticles @ 100 ppm for 15 min | 96.67 ± 3.33 a | 14.89 ± 0.4cde | 14.62 ± 0.26bcd | 2851.5 ± 103.7defg |
| T3: Seed primed with synthesized molybdenum-based nanoparticles @ 150 ppm for 15 min | 100.00 ± 0 a | 16.09 ± 0.74bc | 15.11 ± 0.06abcd | 3119.33 ± 80.08bcd |
| T4: Seed primed with synthesized molybdenum-based nanoparticles @ 200 ppm for 15 min | 100.00 ± 0 a | 16.95 ± 0.58b | 16.05 ± 0.29ab | 3299.67 ± 83.18ab |
| T5: Seed primed with synthesized molybdenum-based nanoparticles @ 250 ppm for 15 min | 93.33 ± 3.33 a | 13.64 ± 0.32defg | 13.55 ± 0.74def | 2539.33 ± 152.5ghi |
| T6: Seed primed with synthesized molybdenum-based nanoparticles @ 300 ppm for 15 min | 93.33 ± 3.33 a | 13.36 ± 0.62efg | 12.78 ± 0.28efg | 2436.43 ± 57.76ghi |
| T7: Seed primed with synthesized molybdenum-based nanoparticles @ 350 ppm for 15 min | 90.00 ± 0 a | 12.63 ± 0.32fgh | 11.81 ± 0.39gh | 2200.2 ± 45.02ijk |
| T8: Seed primed with distilled water for 15 min | 90.00 ± 0 a | 10.89 ± 0.4h | 11.14 ± 0.57h | 1983 ± 17.58k |
| T9: Seed primed with synthesized molybdenum-based nanoparticles @ 50 ppm for 30 min | 93.33 ± 3.33 a | 14.02 ± 0.31def | 14.38 ± 0.12cd | 2650.87 ± 103.5efgh |
| T10: Seed primed with synthesized molybdenum-based nanoparticles @ 100 ppm for 30 min | 96.67 ± 3.33 a | 15.34 ± 0.14bcd | 14.83 ± 0.37bcd | 2913.77 ± 80.36cdef |
| T11: Seed primed with synthesized molybdenum-based nanoparticles @ 150 ppm for 30 min | 100.00 ± 0 a | 16.57 ± 0.31bc | 15.78 ± 0.59abc | 3234.67 ± 73.89abc |
| T12: Seed primed with synthesized molybdenum-based nanoparticles @ 200 ppm for 30 min | 100.00 ± 0 a | 18.84 ± 0.03a | 16.49 ± 0.29a | 3532.67 ± 29.87a |
| T13: Seed primed with synthesized molybdenum-based nanoparticles @ 250 ppm for 30 min | 90.00 ± 5.77 a | 13.28 ± 0.29defg | 12.51 ± 0.24fgh | 2350.7 ± 147.46hij |
| T14: Seed primed with synthesized molybdenum-based nanoparticles @ 300 ppm for 30 min | 90.00 ± 0 a | 12.97 ± 0.89defg | 12.37 ± 0.28fgh | 2340 ± 66.17hij |
| T15: Seed primed with synthesized molybdenum-based nanoparticles @ 350 ppm for 30 min | 90.00 ± 0 a | 12.72 ± 0.11fg | 12.10 ± 0.31fgh | 2234.4 ± 21.04ijk |
| T16: Seed primed with distilled water for 30 min | 90.00 ± 0 a | 11.81 ± 0.2gh | 11.51 ± 0.22gh | 2098.8 ± 22.71jk |
| T17: Seed treatment with ammonium heptamolybdate (2 g kg− 1 of seed) | 96.67 ± 3.33 a | 15.89 ± 0.45bc | 15.03 ± 0.02abcd | 2987.27 ± 98.48bcde |
| SEm ± | 2.56 | 0.40 | 0.37 | 83.19 |
| C.D. at 1% | - | 1.14 | 1.08 | 240.10 |
Values are expressed as mean ± standard error of mean (SEm) of three independent replicates (n = 3). Means followed by the same lowercase letter within a column are not significantly different, while different letters indicate significant differences according to Duncan’s Multiple Range Test (DMRT) at p ≤ 0.01. CD represents critical difference at 1% level of significance
Fig. 7.
Treatment wise comparison of seedling vigour of greengram. (Refer Table 5 for treatment details of T1-T17)
Discussion
The dissolution of ammonium heptamolybdate in water results in its dissociation into ammonium (NH₄⁺) and polymolybdate (Mo₇O₂₄⁶⁻) ions, with the latter being able to partly shift towards the formation of orthomolybdate (MoO₄²⁻) species depending on the pH of the medium:
 |
1 |
The natural electron donors in the form of phenolic hydroxyl and flavonoid catechol groups, which are found in plant extracts, reduce Mo(VI) to lower oxidation states, Mo(IV). At the same time, functional groups like –OH, –C = O and –NH₂ grab the molybdenum atoms, thereby creating a layer that stabilizes the nanoparticles [20]. Through ion–dipole and dipole–dipole interactions between the molybdate ions and the polar phytochemicals, the stability of the colloid is further supported. Moreover, the electrostatic repulsion that hinders the aggregation of particles and the strong coordinate bonding (Mo–O and Mo–N) between the metal centers and donor atoms also contribute to the stability of the system. These interactions work together to produce a stable, bio-capped molybdenum organic complex [21]. The observed bathochromic or hypsochromic shifts (Fig. 3) in λmax (wavelength of maximum absorption) in comparison with the plant extracts and the precursor confirm that the interaction between molybdenum ions and bioactive compounds contributes to the production of stable nanoparticles [22]. The Z-average particle size revealed that the type and amount of phytochemicals in each extract had a major impact on the nucleation and growth of the nanoparticles. The average particle size obtained from SEM analysis (45.20 nm) was larger than the Z-average particle size (17.13 nm) determined by dynamic light scattering (DLS)-based particle size analysis. This difference is expected because the two techniques are based on different measurement principles and sample preparation procedures. DLS determines the hydrodynamic diameter of nanoparticles dispersed in suspension from their Brownian motion, whereas SEM measures the physical dimensions of dried nanoparticles deposited on a substrate. During SEM sample preparation, solvent evaporation and dehydration may promote particle aggregation or agglomeration, resulting in larger apparent particle sizes than those observed in suspension. Therefore, the observed discrepancy reflects the different analytical principles and physical states of the samples rather than inconsistency in nanoparticle formation. The decrease in TPC after nanoparticle synthesis is indicative of the active role played by phenolic compounds in reducing and stabilizing molybdenum-based nanoparticles, which is in agreement with previous studies, whereby polyphenols acted as efficient electron donors during the green synthesis of metal nanoparticles [23]. The drop in TFC is suggestive of involvement of flavonoids in the synthesis of nanoparticles where they play the role of both reducing agent and stabilizer. Previous studies also reported similar results where extracts rich in flavonoids were effective in mediating the green synthesis of metal nanoparticles [23]. Elements associated with phytochemical residues from the Vitex negundo extract acted as capping and stabilizing agents. The absence of any extraneous elemental peaks confirmed the purity and successful formation of molybdenum-based nanoparticles [24]. Seed vigour index significantly increased with molybdenum-based nanoparticles priming, indicating better seedling establishment and metabolic efficiency [25]. This improvement may be attributed to the known role of molybdenum as a cofactor for enzymes such as nitrate reductase and nitrogenase, which are involved in nitrogen metabolism during germination, as reported in previous studies [26–29]. The improved seedling growth observed following nanoparticle priming may be associated with enhanced availability of molybdenum, which could facilitate reserve mobilization and enzyme-mediated metabolic processes during germination, as suggested in previous studies [26–29]. The impact of molybdenum-based nanoparticles on seedling growth was dependent on the concentration. Treatments above 200 ppm brought about a substantial decrease in seed vigour index, shoot length and root length, which may be attributed to the hormetic effect of nanoparticles. Hormesis is characterized by a biphasic response in which low to moderate concentrations stimulate physiological and metabolic activities, whereas higher concentrations exert inhibitory effects. At optimum concentrations, molybdenum-based nanoparticles may promote physiological processes associated with seed germination, including nutrient mobilization, nitrogen metabolism and possible enhancement of antioxidant defence systems. However, these physiological responses were not directly investigated in the present study. The concentrations above 200 ppm may have exceeded the physiological tolerance threshold of seedlings, leading to excessive accumulation of nanoparticles and generation of reactive oxygen species (ROS), which could have potentially induced oxidative stress and cellular damage. Higher concentrations could also have disturbed osmotic balance, impaired water uptake during imbibition, interfered with nutrient absorption and promoted cell wall thickening and lignification, ultimately restricting cell elongation and meristematic activity. The inhibitory reaction at the higher concentrations might be attributed to the phytotoxicity of excessive nanoparticles that may hinder water uptake and metabolic processes during germination. Moreover, molybdenum-based nanoparticles in high amounts might have caused oxidative stress which, in turn, might have caused reduction of accessible dividing and elongated cells [30, 31]. These mechanisms remain hypothetical in the context of the present study because they were not experimentally evaluated but were reported in previous studies. The findings indicated that molybdenum-based nanoparticles showed positive effects at the right dosages but inhibits growth when given more than 200 ppm, which clearly points out the necessity for proper dose adjustment [26, 32]. The present study was limited to laboratory-based seed germination assays. Although the synthesized molybdenum-based nanoparticles improved early seedling growth, further greenhouse and field validation is required before broader agronomic applications can be recommended.
Conclusion
This study successfully demonstrated the green synthesis of molybdenum-based nanoparticles using six plant extracts, providing an environmentally friendly and cost-effective approach for nanoparticle production. Characterization by UV-Vis spectroscopy, particle size analysis, FTIR, SEM, EDX, TPC and TFC confirmed the successful formation of phytochemical-capped molybdenum-based nanoparticles with distinct physicochemical characteristics. The extract of Vitex negundo produced nanoparticles with a comparatively uniform particle-size distribution and lower aggregation among the tested extracts, highlighting the influence of plant phytochemicals on nanoparticle nucleation and stabilization. Seed germination studies on Vigna radiata indicated that priming with synthesized molybdenum-based nanoparticles at 200 ppm for 30 min significantly improved early seedling growth under laboratory conditions. However, these findings should be considered preliminary because the biological evaluation was conducted using a relatively small number of seeds under controlled laboratory conditions. Therefore, additional studies involving larger sample sizes, greenhouse experiments and field trials are required before recommending these nanoparticles as eco-friendly nano-fertilizers for agricultural applications. Further investigations are also needed to evaluate long-term environmental safety, molecular interactions and large-scale applicability of green-synthesized nano-micronutrients.
Acknowledgements
Not applicable.
Authors' contributions
Conceptualization, investigation, methodology- Jayadeva.H.M, Somashekarappa.P.R, Kadalli.G.G and Benherlal.P.S. Data collection, data curation, collection of review literature, original draft preparation, editing- Ritu Dwivedi. Phytochemical analysis- Ritu Dwivedi, Surabhi, Shreedevi Jinnur and K.S.Somashekar. Nano molybdenum synthesis- Ritu Dwivedi and Amina.M. Nano molybdenum estimation – Manoj.K.N and Adarsha.T.S. Characterisation of nano molybdenum- Pruthviraj.N and Shruthi.D.L.
Funding
This research received no external funding.
Data availability
The data supporting the findings of this study are available from the corresponding author upon reasonable request.
Declarations
Ethics and consent to participate declarations
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
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Contributor Information
Ritu Dwivedi, Email: ritudwivedi72@gmail.com.
Jayadeva. H.M, Email: jayadeva98@rediffmail.com
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Associated Data
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Data Availability Statement
The data supporting the findings of this study are available from the corresponding author upon reasonable request.