Eco-friendly synthesis of zinc oxide nanoparticles (ZnOnps) from Piper betel leaf extract: spectral characterization and its application on plant growth parameters in maize, fenugreek and red gram

ABSTRACT

Zinc oxide nano-particles (ZnONPs) were synthesized by the aqueous extract of the betel leaf and characterized by UV-visible spectroscopy, XRD analysis, Zeta potential and SEM studies. The ZnONPs had spherical to irregular shapes ranging from 45.8 to 68 nm with a zeta potential of −22.2 mV. The biogenic ZnONPs are used for plant growth properties in maize and fenugreek. The ZnONPs had more than two to three-fold increase in the plant root, height and yield in the plants. Further, the ZnONPs had no significant changes in levels of antioxidant enzymes in the treated plants but slightly reduced the biofertilizers in the plant rhizospheres and no significant changes in the nodulation of Rhizobium spp. Therefore, the biogenic ZnONPs synthesized exhibited excellent nano-fertilizers in non-nitrogen fixing (maize) and nitrogen-fixing plants (Fenugreek and Red Gram) with multiple modes of fertilization in soil and plants.

KEYWORDS:

• Zinc
• nanoparticles
• plant growth promotion
• biofertilizers effects

Introduction

Nanotechnology is developing rapidly because of the exclusive properties of nanoparticles such as small size, and large surface area to volume ratio [1,2]. Metal and metal oxide nanoparticles have gained interest in recent years because of their applicability in medicine, physics, chemistry and biology. Silver nanoparticles have more valuable properties; silver SPR energy is far from the interband transition energy which other nanoparticles like gold, copper and Pd have less. Silver nanoparticles are employed in clothing, food and cosmetic industries because of their efficient antimicrobial activity [3]. Silver has been confirmed to be an effective antimicrobial agent that has low toxicity in humans [4]. Nanoparticles act as good alternatives to antibiotics because of its efficient antibacterial properties against multidrug-resistant bacteria. Silver nanoparticles can bind to many negatively charged nanoparticles like DNA, RNA and proteins, making them more toxic to the bacteria. Silver nanoparticles exhibit their activity through ROS or interfere in their DNA replication [3]. ZnO nanoparticle is referred to as a multitasking metal oxide because of its wide application use in electronics, industrial, cosmetic, solar energy and healthcare sectors. ZnO is non-toxic and has been recognized as safe by the U.S Food and Drug Administration [5,6]. ZnO nanoparticles are known to play a role in the medical field because they do not cause any toxicity or damage to the DNA of human beings [7,8].

Nano-fertilizers are known to release nutrients to plants and reduce or prevent eutrophication and pollution of the environment. It is also known to reduce the toxic effects of conventional chemical fertilizers. The nano-fertilizers containing zinc oxide nanoparticles (ZnONPs), iron oxide nanoparticles (FeONPs) and copper oxide nanoparticles (CuO-NPs) at ppm levels have increased the crop yield [9]. Among them, ZnONPs are being exploited in various scientific and industrial applications because of their wide band gap and large excitation binding energy [10]. Among the physical and chemical methods of synthesis of ZnONPs, biological methods seem to be highly versatile due to their simplicity, biocompatible and eco-friendly approaches [11,12].

ZnO-NPs are synthesized biogenically utilization of zinc salts that includes zinc nitrite, zinc sulphate and zinc acetate by the plant extract of Calatrophisgiganta leaves [13], Acalypha indica leaves [14]; Parthenium hysterophorus leaves [15]; Hibiscus rosasinensis leaves [16], Azardirachta indica leaves [17], Punica granatum peel extract [18]; Murrayakoengii leaves [19], Camelliasinesis leaves, Olea Europa, Astragalus gummifer root & shoot extract and Citrus aurantifolia. In this present research work, we have biologically synthesized and characterized the ZnONPs using betel leaf extract and evaluated their plant growth and compatible standards with biofertilizers.

Materials and methods

Betel leaf collection and extraction of metabolites

Betel leaves were collected from the betel yard maintained at IIHR, Bengaluru, India. The leaves were washed in distilled water and air-dried in shade for 1 week. The air-dried leaves were powdered finely and stored in a glass bottle. The 10% (w/v) of betel leaf aqueous extract was prepared in water and boiled for 30 min in low flame. After this, the extract was collected in a glass bottle through filtration at room temperature [20].

Synthesis of ZnOnps

To 1 ml solution of zinc nitrate (ZnNO₃), 100 µl of 1% of aqueous extract of betel leaf was added. The reaction mixture was kept in a shaker maintained at 150 rpm at room temperature for 48 hrs until the formation of nanoparticles [21].

Characterization of ZnOnps

ZnONPs formation was monitored by colour change and followed UV-Vis spectrophotometer. Around 2 mg of ZnONPs was mixed with KBr to prepare the pellet for FTIR analysis. The FTIR spectrum was recorded in the wavelength of 400–4000 cm⁻¹ with a resolution of 1 cm⁻¹ using the FTIR spectrophotometer (Brucker IR Affinity, Japan). The crystalline size and purity of ZnONPs were characterized by an XRD diffractometer [8].

Plant-growth experiments

Fertile red soil of agricultural fields was used for potstudy and NPK content was analysed. The seeds were purchased from Gandhi Krishi Vignan Kendra (GKVK), Bangalore. The seed was coated with respective concentrations (0, 1, 2 and 5 ppm) of ZnONPs. 0.250 g of soil was taken in the small pot (10 cm height, 12 cm deep and 5 cm diameter) in triplicates. Experiments were sowed (maize − 10; Red gram − 10 and fenugreek- 100) in the respective labelled pots. During the experimental study, 60–65% of water was maintained and harvested after 15 days of the time. The germination rate was calculated from the number of germinated seeds by the total seed sowed data. The average germinated seeds, root length, shoot length, root dry weight, shoot dry weight and number of leaves of plants from a triplicate of three independent experiments were recorded. The fresh leaf was collected for estimation of lipid oxidation (Malondialdehyde; MDA), SOD (Superoxide Dimutase) and relative water content (RWC) [22].

Effect of ZnONPs on biofertilizers strains

To test the effect of synthesized ZnONPs on the biofertilizers strain, we prepared in vitro viability test using the Pseudomonas fluorescens strains and in vivo test on the rhizobium colonization using the legume plant, fenugreek. For in vitro testing, the Pseudomonas fluorescens was seeded in the nutrient agar plate and the different concentrations of ZnONPs (0, 1, 2, 5 ppm and controls with 100 µg/ml and 10 µg/ml of betel extracts were used) loaded discs were placed on top of the agar. The plates were incubated at 37°C for 24 h and the inhibition of Pseudomonas fluorescens was recorded. For in vivo testing, Rhizobium spp was inoculated in the soil and fenugreek seeds coated with the ZnONPs were sowed. The plants were harvested after 25 days, and the roots were analysed for the nodulation of rhizobium [23,24].

Antibacterial activity

ZnONPs synthesized from betel leaves were investigated for their antibacterial activity against Pseudomonas fluorescens. The zone of inhibition was identified for this activity using Mueller Hinton agar (MHA). MHA was made and sterilized at 120 lbs for 45 minutes. The sterilized plates were filled with media, which was then allowed to stabilize before solidifying. The test microorganism was swabbed after the wells were cut with the appropriate cutter. Different concentrations of ZnONPs were added, and the plates were then incubated for 24 hours at 37°C. The zone of inhibition was assessed following the incubation period [25]. Ciproxacin (5 μg/ml) was used as a standard for antibacterial tests.

Statistical analysis

Categorical variables were presented as continuous variables as mean ± standard deviation (SD). ANOVA was used to calculate statistically significant differences between the standard and extract tested. Statistical analyses were performed using SPSS package version 21 (SAS Institute Inc., Cary, NC, U.S.A.) [26].

Results and discussion

Synthesis of ZnOnps

As previously mentioned, several factors, including the solid-to-liquid ratio, extraction time, and extracted temperature, had an impact on the extraction yield. The extraction yield of an aqueous-based solvent Piper betle extract was shown to be greater in 2015 by Foo and colleagues [27] compared to an ethanol-based solvent extract. Water was therefore employed as a solvent in this investigation. The reduction of zinc from pale yellow to white precipitate indicated the synthesis of ZnONPs which is in line to the earlier conducted study [28].

Characterization of ZnOnps

UV-visible spectroscopy and X-ray diffraction

The UV-Vis spectra showed the characteristics peak of ZnONPs with an absorbance maximum of 355 nm correlating the ZnONPs synthesized as in an earlier report [29]. XRD analysis of ZnONPs showed 2ɵpeaks value of 32.25°, 34.88°, 36.70°, 47.97°, 56.99°, 63.23°, 66.79°, 68.31°, 69.43° and 77.39°Confirming the ZnONPs synthesised (Figure 1). The spherical phase of zinc oxide is responsible for the peaks (JCPDS 47–1253). Due to the leftovers of the leftovers from the plant extract, there were still some impurity peaks. 10 nm was the crystallite size of NPs determined by Scherrer’s equation. The XRD peaks obtained correlated with the earlier study reported [28].

Figure 1. XRD analysis of ZnONPs synthesized from betel leaf extract.

Scanning electron microscope analysis

The ZnONPs had spherical to irregular shapes ranging from 45.8 to 68 nm (Figure 2). The SEM results indicate that the size and shape of ZnO NPs were influenced by the extract volume. Particle size decreased and a distinct shape developed as the leaf extract volume increased [30,31]. In addition, it was evident that the aggregation caused by the pH at 5–7 existed. ZnONPs’ approximately spherical shapes could be attributed to the compound’s functional groups in the extract [32].

Figure 2. SEM images of ZnONPs synthesized from betel leaf extract.

Zeta potential

The samples were sonicated for five minutes to determine the particle size distribution and zeta potential of ZnO NPs, and the Malvern Zetasizer was used to measure the results. The zeta potential was recorded to be −22.2 mV indicating the stability of the particles. The heterogeneity distribution of ZnO nanoparticles in the colloidal solution can cause the particles in the zetasizer analysis to grow in size [33].

Evaluation of plant growth promotion of ZnOnps

Synthesized ZnONPs were tested for seed germination in non-nitrogen fixing (maize) and nitrogen-fixing plants (Fenugreek and Red Gram). In maize, the percentage of seed germination was significantly increased to96.5 ± 7.6 in 5 ppm of ZnONPs from 65.4 ± 2.5 in 100 ppm of ZnNO₃ solution. In red gram, the percentage of seed germination was significantly increased to 91.2 ± 4.7 in 5 ppm of ZnONPs from 69.2 ± 1.5 in 100 ppm of ZnNO₃ solution. In fenugreek, the percentage of seed germination was significantly increased to 98.7 ± 1.6 in 5 ppm of ZnONPsfrom 67.5 ± 1.2in 100 ppm of ZnNO₃ solution. The significant increase in the seed germination was dose-dependent of ZnONPs when compared to the bulk precursor of ZnNO₃ (100 ppm) was observed (Figure 3).

Figure 3. Representative pictures of seed germination of maize and red gram with untreated and different concentrations of ZnONPs.

The plant growth parameters such as shoot length and root length for all plants were two to three folds increased in ZnONPs when compared to the bulk precursor of ZnNO₃ (100 ppm) was observed (Table 1). Furthermore, the plant stress proteins such as MDA, SOD levels and peroxidase had no significant changes compared to control. To test compatibility with Rhizobium sp., we grow the ZnONPs coated groundnut and fenugreek seed-coated plants with biofertilizer Rhizobium spp. The results showed there were no significant effects on the nodulations (Figures 4,5 and 6).

Figure 4. Representative pictures on growth of red gram with untreated and different concentrations of ZnONPs.

Figure 5. Representative pictures of growth parameters of groundnut with untreated and different concentrations of ZnONPs.

Figure 6. Representative pictures of fenugreek plant and root nodulation by natural rhizobium with untreated and different concentrations of ZnONPs.

Table 1. Plant growth parameters and antioxidant activity of ZnNO₃ and ZnONPs treated plants.

Concentration of ZnONPs (ppm)	Germination rate (%)	Shoot length (cm)	Root Length (cm)	Total weight (g)	SOD (Unit mg^−1 protein min^−1)	Peroxidase (Unit mg^−1 protein min^−1)	RWC (%)	MDA(mMg/fls)
Maize
100 (ZnNO3) Control	65.4 ± 2.5	10.4 ± 2.4	05.2 ± 0.2	1.32 ± 0.14	1.744 ± 0.62	0.168 ± 0.14	75.6 ± 1.2	0.167 ± 0.02
1	69.2 ± 4.2	14.5 ± 1.5	08.3 ± 0.5	1.94 ± 0.25	1.732 ± 0.71	0.165 ± 0.11	73.2 ± 1.5	0.162 ± 0.03
2	88.3 ± 5.5**	28.5 ± 1.7**	21.2 ± 0.5**	4.52 ± 0.32**	1.623 ± 0.65	0.162 ± 0.15	72.5 ± 1.7	0.155 ± 0.05
5	96.5 ± 7.6**	29.2 ± 2.4**	25.3 ± 2.4**	5.25 ± 0.51**	1.611 ± 0.52	0.155 ± 0.16	71.2 ± 1.8	0.146 ± 0.04
Red gram
100 (ZnNO3) Control	69.2 ± 1.5	11.2 ± 2.1	04.1 ± 0.2	1.55 ± 0.12	1.754 ± 0.31	0.154 ± 0.11	74.2 ± 1.5	0.154 ± 0.03
1	70.3 ± 3.4	15.8 ± 1.7	09.7 ± 0.6	1.98 ± 0.14	1.752 ± 0.25	0.165 ± 0.22	72.3 ± 1.2	0.152 ± 0.02
2	84.5 ± 4.1**	29.7 ± 1.2**	24.7 ± 0.4**	3.22 ± 0.24	1.723 ± 0.54	0.158 ± 0.21	71.4 ± 1.5	0.149 ± 0.03
5	91.2 ± 4.7**	30.5 ± 1.7**	26.8 ± 1.7**	4.95 ± 0.42**	1.695 ± 0.48	0.147 ± 0.18	69.2 ± 1.2	0.136 ± 0.05
Fenugreek
100 (ZnNO3) Control	67.5 ± 1.2	09.5 ± 1.4	05.7 ± 0.3	1.25 ± 0.09	1.452 ± 0.22	0.164 ± 0.13	76.7 ± 1.3	0.174 ± 0.02
1	87.8 ± 1.5**	12.9 ± 1.9	08.7 ± 0.5	1.98 ± 0.04	1.442 ± 0.32	0.158 ± 0.12	75.2 ± 1.2	0.171 ± 0.01
2	97.5 ± 1.7**	18.7 ± 1.8**	18.9 ± 0.2**	3.35 ± 0.05**	1.440 ± 0.21	0.154 ± 0.11	74.5 ± 1.7	0.170 ± 0.02
5	98.7 ± 1.6**	20.5 ± 1.4**	21.7 ± 0.4**	1.25 ± 0.05	1.438 ± 0.20	0.152 ± 0.10	73.6 ± 1.2	0.168 ± 0.03

Experiment was conducted in triplicates (n = 3) and mean ± SD value is recorded.

ZnONP application through seed priming and coating treatments improved plant height and number per plot as well as vegetative growth. This improvement could be explained by zinc’s function in the synthesis of tryptophan, which is a precursor to the phytohormone indole-3-acetic acid [34]. Additionally, ZnO nanoparticles can alter the biosynthesis of gibberellins and cytokinins, two phytohormones that can increase the number of internodes in a plant [34]. Moreover, in the early phases of plant development, increased cell elongation may result in a rise in plant height [35].

Since the treated plants’ yield-attributing characteristics improved, the applied ZnONPs guaranteed a higher yield. An increase in chlorophyll content can lead to better photosynthetic efficiency, which is specifically correlated with higher levels of soluble protein, starch, and dry mass. By priming the seeds and coating them with ZnONPs, fodder maize’s macronutrients (NPK) and micronutrients (Zn) were also increased in the shoot and roots. This might be the result of enhanced root system development, as evidenced by longer and more biomass roots, which would increase nutrient uptake [36].

Antibacterial activity

However, to find the effect of ZnONPs on biofertilizer strain, we performed in vitro antimicrobial assay against the commercial strains of Pseudomonas fluorescens (Figure 7). The experiment was conducted in triplicates (n = 3) and the mean ± standard deviation (SD) values were recorded. The results showed there was no significant effect on the growth of the Pseudomonas fluorescens by ZnONPs. The betal leaf extract exhibited a significant zone of inhibition against Pseudomonas fluorescens in the concentrations of 5 µg/ml and 100 µg/ml, respectively, in comparison to the ciproxacin control.

Figure 7. Antibacterial activity against the Pseudomonas fluorescens, biofertilizer strain.

Conclusion

ZnONPs with a zeta potential of −22.2 mV were synthesized from betel leaf extract and precursor ZnNO₃. The nanoparticles had a diameter of 45.8 to 68 nm. With increased seed germination, shoot height, root length and weight of plants like red gram, fenugreek and maize, the ZnONPs improved the parameters of plant growth. Additionally, Pseudomonas fluorescens and Rhizobium spp., two biofertilizers that are highly compatible with ZnONPs, were found in the plants. Moreover, plants treated with ZnONPs did not exhibit a significant change in the stress level proteins. This biogenic zinc oxide nanoparticle fertilizer appears to be a promising method.

Availability of data and materials

This declaration is ‘not applicable’

Authors’ contributions

Harikrishnan A – Data Collection, Investigation, Writing (Original Draft Preparation)

Ramalingam Balachandar – Investigation, Validation, Resources

VijayKumar Veena – Conceptualization, Investigation, Supervision, Writing (Review and Editing)

Ahmed Nadeem – Investigation, Formal Analysis, Resources

Balajee Ramachandran – Formal Analysis, Visualization

Muthupandian Saravanan – Conceptualization, Investigation, Supervision, Writing (Review and Editing)

Ethical Approval

This article does not contain any studies with human and/or animals performed by any of the authors.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The authors acknowledge and extend their appreciation to the Researchers Supporting Project Number (RSP2023R124), King Saud University, Riyadh, Saudi Arabia.