Characterisation and antifungal activity of silver nanoparticles biologically synthesised by Amaranthus retroflexus leaf extract

ABSTRACT Following the emergence of resistant fungal pathogens, silver nanoparticles (AgNPs) biosynthesized by plants have been recognized as promising tools to combat parasitic fungi. This study evaluated the potency of Amaranthus retroflexus in producing AgNPs, followed by testing their antifungal effects. The AgNPs exhibited a maximum absorption at 430 nm through ultraviolet-visible spectroscopy, while the X-ray diffraction indicated that they were crystal in nature. Fourier transform infrared spectroscopy confirmed the conversion of Ag+ ions to AgNPs due to the reduction by capping material of plant extract. The transmission electron microscope analysis further revealed that the AgNPs were spherical ranging from 10 nm to 32 nm in size. The AgNPs at the concentrations of 50, 100, 200, and 400 μg/mL were applied to the growth of plant, mushroom, and human pathogenic fungi. The 50% minimum inhibitory concentrations (MIC50) against Macrophomina phaseolina, Alternaria alternata and Fusarium oxysporum were observed to be 159.80 ± 14.49, 337.09 ± 19.72, and 328.05 ± 13.29 μg/mL, respectively. However, no considerable inhibition was observed regarding Trichoderma harzianum or Geotrichum candidum. These findings may suggest A. retroflexus as a green solution for biosynthesizing AgNPs with potent antifungal activities against plant pathogenic fungi.


Introduction
Recent studies have suggested various biological sources for synthesising silver nanoparticles (AgNPs), including fungi, bacteria and plant extracts [1][2][3][4]. Among other suitable plant species, Amaranthus spp. have been reported to serve as good biological sources for AgNPs synthesis. Amaranthus spp. are flowering plants that belong to Amaranthaceae family and have long been used for food and medicinal purposes [5]. Few studies have shown that extracts obtained from Amaranthus spp. possess antimicrobial activities. According to the studies conducted so far, A. hypochondricus protein extract possesses potent antifungal activities towards Alternaria alternata, Fusarium solani, Candida albicans, Fusarium oxysporum, Trichoderma sp. and Aspergillus ochraceus [6].
In addition to plant extracts, the antimicrobial potential of AgNPs biosynthesised by Amaranthus spp. has been investigated. A recent study reported green synthesis of AgNPs using an aqueous solution of silver nitrate and A. gangeticus Linn (Chinese spinach) leaf extract. The biosynthesised AgNPs were demonstrated to exert antimicrobial activities towards Bacillus subtilis, Shigelle flexineri and Sclerotinia sp [7]. Another study reported fabrication of an Ag-polyvinyl alcohol (Ag/PVA) nanocomposite using A. tristis-synthesised AgNPs. Pseudomonas fluorescens and Klebsiella pneumoniae were found to be inhibited by the Ag/PVA nanocomposite membrane [8].
To the best of our knowledge, no study has reported the biosynthesis of nanoparticles by Amaranthus retroflexus. In addition, there is limited quantified data regarding the susceptibility of parasitic fungi to AgNPs synthesised by Amaranthus spp. Here, we report the design and development of a green method to biosynthesise AgNPs employing dried leaves extracts of A. retroflexus. Further, antifungal activities of these synthesised AgNPs were quantifiably evaluated against several plant, mushroom and human parasitic fungi.

Chemicals
Potato dextrose agar (PDA) media and silver nitrate salt (AgNO 3 , 99%) were purchased from Quelab (Canada) and Merck (Darmstadt, Germany), respectively. All the reagents utilised in this study were freshly prepared before use.

Plant and fungi samples
Seeds of A. retroflexus were provided and authenticated by the Herbarium of Agronomy Department, Faculty of Agriculture, Ferdowsi University of Mashhad, Iran. The seeds were cultivated and the resulting fresh leaves were then collected to be used for further experiments. The following fungal strains were kindly gifted from and authenticated by the Culture Collection of Plant Protection Department, Faculty of Agriculture, Ferdowsi University of Mashhad, Iran: Macrophomina phaseolina, A. alternata, F. oxysporum, Trichoderma harzianum and Geotrichum candidum. The fungi were grown onto PDA and incubated at 25 C to obtain pure cultures.

Preparation of leaf extract
The fresh leaves of A. retroflexus were washed several times in running tap water and finally in distilled water. Four grams of the air-dried leaves were mixed with 100 mL of distilled water, keeping in a water bath at 55 C for 15 minutes followed by cooling to the room temperature. The extracts were then filtered through Whatman filter papers No. 1 and stored at 4 C until use [4].

Biosynthesis of silver nanoparticles by A. retroflexus
A total of 10 mL of the A. retroflexus leaf aqueous extract was added to 90 mL of silver nitrate solution (1 mM). The reaction was allowed to stand in at room temperature overnight, kept in the dark in order to minimise photo activation of silver nitrate. Then, the solution containing the formed AgNPs was centrifuged at 15,000 rpm for 10 minutes followed by drying in a vacuum oven at 60 C for 24 hours [4]. The pellet was collected and re-dispersed in glass-distilled water, removing any interactive biological molecules.

UV-visible spectroscopy
The formation of AgNPs in the leaf extract was confirmed through a colour change from pale yellow to dark brown. The colour change was recorded under the UV-visible (UVvis) spectroscopy between 300 and 700 nm using an Agilent 8453 spectrophotometer (USA).

Transmission electron microscopy
Transmission electron microscopy (TEM) was performed by a Leo 912 AB instrument (Germany). In brief, a drop of appropriately diluted sample of AgNPs was poured on carbon-coated copper grids and allowed to stand for 2 minutes. The excess solution was removed using a blotting paper and allowed to be dried at room temperature.

X-ray diffraction analysis
The lyophilised AgNPs coated on X-ray diffraction (XRD) grid were subjected to XRD measurements. The analysis was carried out in an X-ray diffractometer with an operating voltage of 45 KV and current of 0.8 mA (Unisantis XMD-300, Swiss). The diffraction patterns were recorded by Cu-Ka radiation of wavelength 1.54 A in the region of 2u from 30 to 80 .

Fourier transform infrared spectroscopy
The aqueous leaf extract and AgNPs were subjected to Fourier transform infrared (FTIR) spectroscopy (Thermo Nicolet AVATAR 370) in order to analyse their spectra. The analysis was carried out with KBr pellets, recorded in the range of 500-4000 cm ¡1 .

Antifungal activity of AgNPs
The antifungal potential of the biosynthesised AgNPs was investigated according to a modified method described elsewhere [9]. Five millilitre of the AgNPs at various concentrations (50, 100, 200 and 400 mg/mL in sterile distilled water) was added into 5 mL of the autoclaved media before it solidified. The mixture was poured into the sterile 8-cm Petri dishes. Negative controls containing media only were also considered. The Petri dishes were then incubated in the dark at 25 C for 48 hours, after which the Petri dishes were inoculated with agar plugs of the growing fungal mycelia (5 mm in diameter). The plates were incubated in the dark at 25 8C for further five days. The radial growth of fungal mycelia was calculated using the mean of two fungal colony diameters at right angles. The inhibition potency of AgNPs towards each fungal strain was calculated using the following equation: Inhibition rate (per cent) D R ¡ r/R £ 100; where R is the radial growth of fungal mycelia in the negative control (cm) and r is the radial growth of fungal mycelia challenged with the AgNPs.

Statistical analysis
All the experiments were independently repeated at least three times. In each experiment and for each fungal strain, three Petri dishes were used to calculate the radial growth rate. GraphPad Prism version 6 was utilised to conduct statistical analyses and one-way analysis of variance (ANOVA) tests. Means were compared using Tukey multiple comparisons test with a significance level of 0.05. The 50% minimum inhibitory concentration (MIC 50 ) of the AgNPs was interpolated using linear regression analysis of the relevant dose-response curve.

UV-vis spectroscopy
In this study, dried leaves of freshly growing A. retroflexus plants ( Figure 1) were utilised for extraction. Following the addition of silver nitrate to the dried leaf extract, a colour change from pale yellow to dark brown was observed ( Figure 2). This colour change has previously been attributed to the excitation of surface plasmon resonance by AgNPs [10]. The reduction of AgNPs in the aqueous A. retroflexus leaf solution was further confirmed by the UV-vis spectroscopy. After 24 hours of incubation, the sharpening of the absorption at 430 nm confirmed that the particles were mono-dispersed ( Figure 3).

Transmission electron microscopy analysis
Microscopic features of the AgNPs, including morphology and particle size, were assessed through TEM analysis. Figure 4 illustrates the TEM image of AgNPs synthesised by the leaf extract of A. retroflexus. As depicted by the TEM image, the particles were mostly spherical in shape with a diameter ranging from 10 to 32 nm (Figure 4). This size range is smaller than those of AgNPs synthesised by many other Amaranthus species, including A. viridis; 10-45 nm [11], A. tristis; 20-40 nm [8] and A. dubius; 10-70 nm [12]. By contrast, the size range of AgNPs biosynthesised by A. gangeticus was reported to be 11-15 nm which was smaller than that reported by us here [7]. It has been known that reducing the size of nanoparticles may enhance their antibacterial activities [13]. However, no comparison could be made here with regard to antifungal activity, as any of the aforementioned studies have not reported quantified antifungal activities of AgNPs.   of silver as a face-centred cubic structure [14]. Therefore, the XRD data clearly demonstrated the presence and crystal structure of the silver in the A. retroflexus leaf extract.

Fourier transform infrared spectra
Various absorption bands in the FTIR spectra showed different chemical groups in the extract containing biosynthesised AgNPs. While a broad band at 3273 cm ¡1 showed the stretching vibrations of ¡N¡H and ¡O¡H groups, the absorption bands at 2924, 1640, 1383 and 1033 cm ¡1 corresponded to ¡C¡H, CDO, CDC and C¡O¡ groups, respectively. Additionally, the absorption bands of 1155 and 1068 cm ¡1 confirmed the presence of C¡N group. The weak bands at 726 and 612 cm ¡1 were due to the out-of-plane bending vibrations of ¡O¡H and C¡H groups, respectively. In addition, the peaks recorded  at 1234 cm ¡1 indicated that the amino groups were partially utilised for the encapsulation and stabilisation of the biosynthesised AgNPs ( Figure 6).
The presence of ¡N¡H, ¡O¡H, CDC and ¡C¡H groups in the FTIR spectra suggests that the A. retroflexus extract contained the hydroxyl and amino groups substituted flavonoids, as similarly reported elsewhere [4]. The flavonoids can act as reducing agents (which reduce AgC to Ag0), whereas the amino group serves as a stabilising agent in the green synthesis of AgNPs [15]. Thus, the FTIR spectra obtained in this study may explain the interaction of the leaf biomolecules of A. retroflexus with silver nitrate, leading to biosynthesis of AgNPs. In addition, the FTIR data reveal the multi-functionality of the aqueous extract of A. retroflexus where reduction and stabilisation occur simultaneously.

Antifungal activity of Amaranthus retroflexus-synthesised AgNPs
The inhibition of the mycelia growth by the AgNPs at various concentrations were daily evaluated and photographed for all the tested fungi, in comparison to the negative control. As an example, Figure 7 illustrates the mycelial growth of M. phaseolina challenged with the biosynthesised AgNPs in comparison to the negative control (Figure 7). Moreover, the antifungal activities of the AgNPs were quantified and statistically analysed ( Table 1).
The quantified findings showed differences in antifungal activities of the AgNPs across the tested fungi and various concentrations. The AgNPs significantly prevented the growth of M. phaseolina, A. alternata and F. oxysporum in a dose-dependent manner as compared to the negative control (p < 0.05). Among the tested fungi, the highest  Note: Values represent amounts of prevention (percentage) of fungal mycelial growth as compared to the negative control. Superscript lower-case letters within each row indicate statistical comparisons among the concentrations of each fungus. In the column of the interpolated MIC 50 , superscript upper-case letters show statistical comparisons made between the fungi. Means followed by the same letters are not significantly different (Tukey, p value < 0.05). Nd: not determined because no measurable growth inhibition was observed; MIC 50 : the 50% minimum inhibitory concentration was defined as the concentration of AgNPs at which the mycelial growth of the tested fungi was inhibited as much as 50%.
inhibition rate (71.09% § 1.41%) was observed with M. phaseolina at 400 mg/mL of the AgNPs while the lowest one (9.84% § 2.81%) was seen with F. oxysporum at 50 mg/mL of the AgNPs (p < 0.05). In addition, the least effective concentration of the biosynthesised AgNPs was found to be 50 mg/mL which caused 31% inhibition of the growth in M. phaseolina. The antifungal effect of the AgNPs was very limited against T. harzianum while no measurable antifungal effect was seen with G. candidum even at the highest concentration of the AgNPs (Table 1) According to all of the observational and quantified findings, the AgNPs were found to be much more effective against the growth of plant pathogenic fungi (M. phaseolina, A. alternata and F. oxysporum) than that of human (G. candidum) or mushrooms (T. harzianum) pathogenic fungi. In this regard, M. phaseolina and G. candidum were the most sensitive and resistant microorganism to the A. retroflexus-synthesised AgNPs, respectively.
Due to the environmental concerns regarding the use of chemical agents, bio-control agents are largely used in plant disease control programmes [16]. Among others, several species of Trichoderma have been suggested as promising biological control agents against a number of plant pathogenic fungi, including F. oxysporum [16][17][18]. F. oxysporum is known as a root-infecting fungal pathogen that causes several plant diseases on a broad range of plant species. The findings of our study may have implications in bio-control disease programmes, as no inhibition was observed against T. harzianum even at the highest concentration while the AgNPs considerably inhibited the growth of F. oxysporum. These results may propose that the use of biosynthesised AgNPs along with T. harzianum could serve as effective green tools in order to inhibit the growth of pathogenic fungi F. oxysporum.

Conclusion
In the absence of knowledge on the potency of A. retroflexus in biosynthesising AgNPs, the present study reports the biosynthesis of AgNPs by A. retroflexus leaf extract, confirmed by XRD, TEM, UV-vis spectroscopy and FTIR analyses. Antifungal activities of the biosynthesised AgNPs were then evaluated towards plant, mushroom and human pathogenic fungi. There is a body of information on antimicrobial activities of AgNPs biosynthesised by a broad range of plant species [19], but little attention has been given to their antifungal activities. In conclusion, the data presented here demonstrate that A. retroflexus may be considered a green tool for synthesising AgNPs with efficient antifungal activity, particularly against plant fungi: M. phaseolina and F. oxysporum. Thus, the findings of this study could be adopted for several applications in the plant protection field. Further studies are underway to investigate treatment of pathogenic fungi-damaged plants by the A. retroflexus-derived AgNPs. However, further research is also warranted to investigate whether the application of AgNPs into the soil might cause unwanted damages to useful bacteria, as it has been well known that nanoparticles have potent antibacterial activity. Other biological potentialities, namely antibacterial and anticancer activities of A. retroflexus-mediated nanoparticles could be also studied in the future.