Plant‐mediated green synthesis of silver nanoparticles using Trifolium resupinatum seed exudate and their antifungal efficacy on Neofusicoccum parvum and Rhizoctonia solani

Abstract

In recent years, biosynthesis and the utilisation of silver nanoparticles (AgNPs) has become an interesting subject. In this study, the authors investigated the biosynthesis of AgNPs using Trifolium resupinatum (Persian clover) seed exudates. The characterisation of AgNPs were analysed using ultraviolet–visible spectroscopy, X‐ray diffraction (XRD), transmission electron microscopy (TEM) and Fourier transform infra‐red spectroscopy. Also, antifungal efficacy of biogenic AgNPs against two important plant‐pathogenic fungi (Rhizoctonia solani and Neofusicoccum Parvum) in vitro condition was evaluated. The XRD analysis showed that the AgNPs are crystalline in nature and have face‐centred cubic geometry. TEM images revealed the spherical shape of the AgNPs with an average size of 17 nm. The synthesised AgNPs were formed at room temperature and kept stable for 4 months. The maximum distributions of the synthesised AgNPs were seen to range in size from 5 to 10 nm. The highest inhibition effect was observed against R. solani at 40 ppm concentration of AgNPs (94.1%) followed by N. parvum (84%). The results showed that the antifungal activity of AgNPs was dependent on the amounts of AgNPs. In conclusion, the AgNPs obtained from T. resupinatum seed exudate exhibit good antifungal activity against the pathogenic fungi R. solani and N. Parvum.

1 Introduction

Silver nanoparticles (AgNPs) are particles in the size of 1–100 nm and they contain 20–15,000 silver atoms [1]. AgNPs due to the special unique physical, chemical, biological and environmental properties have attracted considerable interest. These particles are widely used in research, environment, agriculture and industries such as water purification [2], packaging [3], textile engineering [4], medicine (treating wounds and burns) [5], formulation of dental resin composites [6], medical devices and implants [7, 8], cosmetics [9] and as antimicrobial agents [10, 11]. Several studies have been carried out widely on the antimicrobial properties of AgNPs and their effects against various pathogens such as viruses, fungi and some bacteria species [5, 12, 13]. Most of them have confirmed the antimicrobial properties of AgNPs.

The production of AgNPs is performed by variety of chemical and physical methods. Synthesised nanoparticles in the applied procedures are unstable and toxic. The high costs and the environmental hazards are some of the disadvantages of these methods [14]. In recent years, the use of biological methods with plants and plant materials and microorganisms (bacteria, fungus, algae and enzymes) for the production of AgNPs has been of interest [15, 16, 17, 18]. The AgNPs in biological synthesis have properties such as non‐toxicity, environmentally friendly, simplicity, cost effective and it also provides efficacious single‐pot reactions without additional surfactants or capping agents which were suggested as possible eco‐friendly alternatives to the chemical and physical methods [4, 19].

Persian clover (Trifolium resupinatum L.) is an annual leguminous forage well grown in semi‐arid conditions of Mediterranean areas [20]. Rhizoctonia solani is a very common soil borne pathogen with a great variety of plant hosts [21, 22]. This fungi can attack wide range of plant species such as beans causing seed decay and damping off, hypocotyl rot, root rot and web blight [23]. Neofusicoccum parvum is a causal agent for canker symptom of Eucalyptus species in many parts of the world [24]. This could cause the fruit rot and shoot the blight of the peach trees [25]. This study was aimed to synthesise the AgNPs using T. resupinatum seed exudates and to evaluate in vitro their antifungal efficacy on N. parvum and R. solani.

2 Material and methods

2.1 Reagents and fungal strains

Silver nitrate (AgNO₃) was purchased from Merck Company (Darmstadt, Germany). Seeds of T. resupinatum were obtained from Pakan‐bazr Co., Isfahan, Iran. In Fig. 1, the plant and seeds of T. resupinatum are shown. A voucher specimen of the seeds of T. resupinatum was deposited at the Herbarium of Department of Pharmacognosy of School of Pharmacy, Kerman University of Medical Science, Kerman, Iran (KF1609). Fungal strain R. solani (AG2_2) and N. parvum were obtained from the Department of Plant Protection, University of Kerman, Iran. R. solani (AG2‐2) and N. parvum were subcultured on potato dextrose agar (PDA) (Merck, Darmstadt, Germany).

Fig. 1.

Plant and seeds of T. resupinatum

a Plant

b Seeds

2.2 Preparation of T. resupinatum seed exudates

About 10 g of Persian clover (T. resupinatum L.) seeds were disinfected for 5 min in 2% sodium hypochlorite. The seeds were washed three times with distilled water (DW) to eliminate the sodium hypochlorite. Then, the seeds were placed in 70% alcohol for 2 min and then rinsed four times with sterile DW then imbibed in deionised water (DI) (1 g dry weight/10 ml DI water). Following seven days of incubation at 28°C in darkness, the seeds were removed from the soaking medium [26, 27]. The supernatant phase was collected and liquid fraction from any large insoluble particles was separated by centrifugation at 3000g (4500 rpm) for 20 min, then filtered with Whatman filter paper No. 1. During the experiment, exudates pH was 4.5.

2.3 Biosynthesis of AgNPs

About 15 ml of the obtained seed exudates was diluted by 30 ml sterile DI and different concentrations of AgNO₃ (1, 2.5, 4 and 5 mM) was added to the mixture in order to reduce Ag⁺ to Ag⁰ [28]. AgNO₃ was not added to the control. The reaction mixture was stored at 28°C in the dark for 1, 7, 30 and 60 days. With the synthesis of AgNPs, the colour solution was converted from colourless to dark brown. The AgNPs formation from T. resupinatum seed exudate was evaluated by ultraviolet–visible (UV–Vis) spectrophotometer analysis.

2.4 Characterisations of AgNPs

2.4.1 UV–Vis spectroscopy

The biosynthesis of AgNPs was choked periodically by UV–Vis spectrophotometer (Scan Drop‐type product, Analytik Jena, Germany) in room temperature. The absorption wavelength was read at different concentrations and times in a range between 300 and 700 nm [29, 30]. The colour of T. resupinatum seed exudates solution was converted from colourless to dark brown after 1, 7, 30 and 60 days of the reaction. This colour change is due to the AgNPs formation. Maximum absorbance of the UV–Vis spectra was seen at 428 nm which increased with time of incubation of AgNO₃ with the T. resupinatum extract. The curves presented the increase in absorbance at various time intervals (1, 7, 30 and 60 days) and the peaks at 428 nm corresponding to the surface plasmon resonance of AgNPs are observed. The observation demonstrated that the reduction of the Ag+ ions was accrued [31].

2.4.2 X‐ray diffraction (XRD)

In this study, we studied the formation and quality of compounds using the XRD technique. For this purpose, the biosynthesised AgNPs colloid was centrifuged (at 15,000 rpm; 25°C) for 10 min, washed with DI water and centrifuged again for four cycles. Then, the purified AgNPs were dried at room temperature and subjected to the XRD experiment. The XRD was performed on an X‐ray diffractometer (X'Pert Pro MPD). The scanning was performed in the region of 5θ from 20° to 80° [26].

2.4.3 Analysis of Fourier transform infra‐red spectroscopy (FTIR)

For FTIR analysis, the powder sample of AgNPs was prepared by centrifuging the synthesised AgNPs solution at 6000 rpm for 10 min. The solid residue obtained is washed with DI water for removing any unattached biological moieties from the surface of the nanoparticles, which are not responsible for biofunctionalisation and capping. The resultant residue is then dried completely by incubating at 60°C for 48 h, and the powder obtained is used for FTIR measurements carried out by the KBr pellet method using Bruker – Model TENSOR II – FTIR spectrometers (Germany) instrument in the range of 400–4000 cm⁻¹ at a resolution of 4 cm⁻¹ at room temperature [32, 33].

2.4.4 Transmission electron microscopy (TEM)

The morphology of the AgNPs includes the size, shape, morphology and distribution of the formed AgNPs which were determined by using TEM (Carl ZIESS Microscope, Germany). The size distribution pattern was investigated from observing the SEM images by measuring the diameters of at least 100 particles.

2.5 Antifungal activity of AgNPs

In this study, we assayed the inhibitory effect of various concentrations of synthesised AgNPs (0, 2.5, 5, 10, 20 and 40 ppm) on the mycelia growth of R. solani and N. Parvum. For this purpose, an in vitro assay was performed on PDA medium treated with different concentrations of AgNPs (0, 2.5, 5, 10, 20 and 40 ppm). Various concentrations of AgNPs were poured to PDA medium prior to plating in 75×15 mm Petri dish. The media that contained various concentrations of AgNPs was incubated at 25 ± 2°C. At 24 h after the inoculation, agar plugs of uniform size comprising of fungus were inoculated at the centre of each Petri dish containing AgNPs. The PDA plates were incubated at 28 ± 2°C for 4 and 3 days to allow the growth of R. solani and N. parvum, respectively. When the control plate was covered completely with fungal mycelial growth, the radial growth of fungal mycelium was measured [34]. Each treatment was replicated three times and the experiment was repeated twice. The percentage of the inhibition of growth rate was calculated via the following equation: (the radial growth of the fungal mycelia on the plate which was treated with AgNPs was subtracted from the radial growth of the fungal mycelia on the control plate/the radial growth of the fungal mycelia on the control plate)×100

$$\mathrm{Inhibition}\mathrm{rate}=\frac{R-r}{R}\times 100$$

where R is the radial growth of the fungal mycelia on the control plate and r is the radial growth of the fungal mycelia on the plate containing AgNPs.

2.6 Data analysis

By using the analysis of variance, the data which were gathered from the tests was analysed via using SAS ver. 9.2 and the means were compared with the Duncan's multiple range tests (DMRT). In the analysis, if the comparison between the positive control and the medium treated with AgNPs showed P value < 0.05, this was considered to be statistically significant.

3 Results

3.1 UV–Vis absorbance studies

The colour change of the T. resupinatum seed exudates from colourless to dark brown is due to the AgNPs formation. This indicates the reduction of Ag⁺ to Ag⁰ during exposure to the T. resupinatum seed exudates (see Fig. 2). The UV–Vis spectra of biosynthesised AgNPs by T. resupinatum seed exudates are shown in Fig. 3 at different times and various concentrations. In the UV–Vis absorption spectrum, a strong and broad peak located at 428 nm was observed.

Fig. 2.

Colour change of the T. resupinatum seed exudates solution containing silver before and after synthesis of AgNPs. a: T. resupinatum seed exudates solution. b, c, d, e: T. resupinatum seed exudates solution containing 1, 2.5, 4, 5 ppm of synthesised AgNPs, respectively

Fig. 3.

UV–Vis spectra of biosynthesised AgNPs by T. resupinatum seed exudates

a At different times

b At various concentrations

3.2 XRD analysis

The analysis of XRD pattern shows five intense peaks in the whole spectrum of 2θ values ranging from 20 to 100. These intense peaks were observed at 2θ values of 38.098°, 44.154°, 64.67°, 77.544° and 83° which correspond to (111), (200), (220), (311) and (331) respectively (see Fig. 4). Thus, the XRD pattern clearly shows that the AgNPs was formed by the reduction of Ag⁺ by T. resupinatum seed exudates. The nature of AgNPs is crystalline. All peaks in the XRD pattern can be readily indexed to Bragg's reflection based on the face‐centred cubic structure of AgNPs [33].

Fig. 4.

XRD pattern of AgNPs synthesised by treating AgNO₃ solution with T. resupinatum seed exudates

3.3 FTIR analysis

Fig. 5 shows FTIR spectra obtained from the powder made from synthesised AgNPs by using T. resupinatum seed exudates. The FTIR study indicates that the carboxyl (−C=O), hydroxyl (O−H) and amine (N–H) groups in the Persian clover seed exudates are mainly involved in the reduction of Ag⁺ ions to Ag⁰ nanoparticles. In FTIR spectrum of the biosynthesised AgNPs, five absorption bands were observed at 3429, 2928, 1632, 1406, 1103 and 617 cm⁻¹. The strong absorption band at 3429 cm⁻¹ in the FTIR spectra were assigned to stretching vibrations of H in O–H, in alcohols and phenols and, in some degree to N–H stretching vibrations in amines. The band at 2928 cm⁻¹ corresponds to the asymmetric stretching of the bonds [35].

Fig. 5.

FTIR spectra recorded from powder of the synthesised AgNPs using T. resupinatum seed exudates

The peak located at 1631 cm⁻¹ clearly indicates the C–O stretching in carboxyl or C–N bending in the amide group. A shift in this peak (from 1631 to 1632 cm⁻¹) showed the possible involvement of the amino or carboxyl groups of the seed exudate in nanoparticle synthesis. The vibration shift around 1445–1454 cm⁻¹ was indicative of the involvement of aliphatic and aromatic C–H plane deformation vibrations of methyl, methylene and methoxy groups in the reductive process [32]. The band at 1632 cm⁻¹ demonstrates the fingerprint region of the CO, C–O and O–H groups, and the bands at 1632 and 1406 cm⁻¹ were assigned for aliphatic amines.

3.4 TEM analysis

TEM is useful to determine the morphology (size and shape) of the AgNPs. The TEM images of the prepared AgNPs in two different scales of 25 and 50 nm are observed in Fig. 6 a. TEM images showed spherical Ag particle structures. The particle size distribution histogram determined by TEM is shown in Fig. 6 b. An AgNPs nanoparticle ranges in size from 1 to 25 nm. The maximum particle size distributions in the diameter range ub‐10 (5–10) nm.

Fig. 6.

TEM images and histogram of AgNPs

a TEM images of AgNPs in different scales

b Histogram of particle size distribution of the AgNPs

3.5 AgNPs and their antifungal activities

In this study, we assayed inhibitory effects on PDA agar plates supplemented with various concentrations of synthesised AgNPs (0, 2.5, 5, 10, 20 and 40 ppm) on growing R. solani and N. Parvum. The inhibition effect of AgNPs at different concentrations was analysed. The highest inhibition effect was observed against R. solani at 40 ppm concentration of AgNPs (94.1%) followed by N. parvum (84%). R. solani isolate was more sensitive than that of N. parvum at 40 ppm concentration of AgNPs. The lowest antifungal activity was observed against N. parvum at 2.5 ppm concentration of AgNPs (Table 1). The inhibitory effects of various concentrations of synthesised AgNPs (0, 2.5, 5, 10, 20 and 40 ppm) on the mycelia growth of R. solani and N. Parvum are shown in Table 1. In addition, greater inhibition than 90% was observed against two fungi on PDA media which were treated with a 40 ppm concentration of AgNPs. The inhibition effect of AgNPs against N. parvum and R. solani on PDA in vitro is shown in Fig. 7 . Furthermore, in Table 2 the colony diameter of two tested fungal strains (mm) at different concentrations of AgNPs is shown. We demonstrated that the inhibitory effect increases with increasing concentration of AgNPs.

Table 1.

Inhibitory rate (%) of AgNPs at various concentrations (ppm) against R.solani and N. Parvum on PDA media in vitro

Strains	Inhibition rates (%)a in various concentrations
	2.5 ppm	5 ppm	10 ppm	20 ppm	40 ppm
R. solani	56	67	80	85	91
N. parvum	15	63	71	73	84

Means followed by a different letter(s) in the same column differ significantly (p = 0.05) according to DMRT

^a Inhibition rates were determined based on five replicates of each experiment, and the inhibition rate of control = 0

Fig. 7.

Inhibition effect of AgNPs against

a N. parvum on PDA in vitro

b R. solani on PDA in vitro

CTR: control

Table 2.

Colony diameter of N. parvum and R. solani on PDA (mm) at various concentrations of AgNPs

Strains	AgNPs concentration, ppm
	0	2.5	5	10	20	40
N. parvum	75(±2.16)	33(±3.15)	25(±2.08)	20.15(±2.18)	20(±2.30)	12.5(±0.67)
R. solani	75(±2.01)	31.4(±2.53)	24.2(±2.66)	15(±2.21)	11.1(±2.26)	6.8(±1.42)

4 Discussion

In recent years, the green approach to synthesis of nanoparticles using plant materials as novel way has gained much interest for chemists and researchers. AgNPs have been synthesised using various plant extracts, including magnolia (Magnolia kobus) leaf broth, coffee and tea extracts, Cassia fistula leaf broth, Eclipta leaf extract [36] and Pterocarpus santalinus [37]. The characterisation of the synthesised AgNPs using various plant exudates vary greatly in their size, stability, shape, morphology and their antimicrobial efficacy.

The characterisation of AgNPs was investigated using UV–Vis spectroscopy, TEM, FTIR and XRD. Then, the inhibitory effects of synthesised AgNPs at various concentrations were assayed against R. solani and N. Parvum. We concluded that synthesised AgNPs using T. resupinatum seed exudates can act as a bio‐reluctant and bio‐capping agent and have the potential to inhibit the growth of R. solani (AG2_2) and N. parvum in in vitro conditions.

AgNPs synthesised by T. resupinatum seed exudates have spherical shape and their size ranges from 1 to 25 nm with an average of 17 nm. Amaladhas and his coworkers [38] had shown that the leaf extract of Cassia angustifolia nanoparticles were poly‐dispersed and spherical in shape with a particle size in the range of 9–31 nm, the average size was found to be 21.6 nm at pH 11. On the other hand, the use of banana, neem and tulsi extracts for AgNPs formation resulted in approximately spherical, triangular and cuboidal AgNPs, respectively [29]. The difference in size, charge, stability, shape and morphology synthesis AgNPs, also in areas of the peaks were obtained in the FTIR analysis, and the XRD pattern referred to the availability of the different quantity and nature of the capping factors present in the various t leaf extracts.

In this work, a strong and broad peak in UV–Vis absorption spectrum in 428 nm was observed. The characteristic peak location in the green synthesis of AgNPs which have been prepared using Sterculia foetida L. young leaves aqueous extract [39] and Geranium leaf [40] represented the AgNPs formation and were similar to our finding. In contrast, reports from Anal K. Jha and Prasad [41] suggested that observing the peak in UV–Vis shows that the absorption spectrum occurs at 449 nm. The difference may correlate with the size and shape of the Cycas leaf negotiated synthesis of the AgNPs. The nanoparticles were found to be 2–6 nm in size and spherical in shape.

Additionally, the inhibitory effects of the AgNPs synthesised by T. resupinatum seed exudates were assayed against R. solani and N. Parvum. The results showed that the green‐synthesised AgNPs have potential antifungal effect. Numerous studies have reported that synthesised AgNPs from plant extracts have inhibitory effects on the pathogenic fungi (yeasts and molds), many bacteria and viruses [42, 43]. The antimicrobial property and toxicity of AgNPs leaf extracts of some Indian plants such as Azadirachta indica (neem), Musa balbisiana (banana) and Ocimum tenuiflorum (black tulsi) against Escherichia coli and Bacillus spp. were demonstrated by Banerjee et al. [29]. They reported that the maximum bacterial inhibition for Gram positive bacillus occurs by the AgNPs synthesised using banana leaf extracts.

The effects of the AgNPs on the phytopathogens fungi with sclerotium forming such as R. solani, Sclerotinia sclerotiorum and S. minor were investigated. Various inhibitory effects of the AgNPs were observed on their hyphal growth of these fungi in the following order: R. solani > S. sclerotiorum > S.minor [44]. In addition, the AgNPs of S. foetida leaves at 80 μl/well displayed maximum antibacterial activity against E. coli, followed by Bacillus subtilis, Staphylococcus aureus and minimum activity against M. gypseum and T. tonsurans [39]. Also, Amaladhas et al. [38] demonstrated that the AgNPs synthesis using Cassia angustifolia (senna) have more antimicrobial efficiency on the growth of S. aureus (22 mm) than that of E. coli (16 mm). In another study, the antibacterial and antioxidant properties of AgNPs using farnesiana (Sweet acacia) seed extract against E. coli and S. aureus followed by B. subtilis and P. aeruginosa have also been reported [45]. Also, their findings indicated that AgNPs have very good antioxidant property. The antimicrobial mechanism of AgNPs has not been cleared well. The inhibitory actions of AgNPs have been referred to the following reasons: upon its binding to cytoplasmic membrane, the cell membrane damages, then the pits form on the cell surface, and cell wall's permeability modifies. In addition, it was shown that AgNPs can inhibit major function of cellular respiration, DNA replication and cell division which results in the loss of cell viability and cell death [37, 46, 47].

Studies by various investigators have suggested that the antimicrobial properties depend on size of the nanoparticles [48, 49]. Smaller size nanoparticles exhibit more antimicrobial activity. The increased antimicrobial activity has been correlated to their small size and larger surface area also provides higher reactivity of particles and more chances of interacting with solvent ions that will draw higher ions from particles [50, 51]. In this work, we synthesised AgNPs with the average size in the range from 1 to 25 nm. The small AgNPs synthesised have promising antifungal potential against R.solani and N. Parvum. As well as, the highest level of inhibition was observed on two phytophatogenic fungi on the media treated with a 40 ppm concentration of AgNPs. We found that the antifungal activity of AgNPs showed a dose‐dependent pattern.

Our result is similar with the findings reported by Min et al. [44] who suggested that increase in AgNPs concentrations leads to increment in the inhibitory effect. Also, the reports from other groups, such as Kim et al. [34], Atta et al. [52] and Singh and Vidyasagar [39] confirmed this result. In higher concentrations of AgNPs, their connection to the fungus walls, the interaction between them and the destruction of fungal pathogens were increased [34].

5 Conclusion

In this study, we successfully used T. resupinatum seed exudates for the synthesis of nanoparticles. Compared with the chemical and physical methods, the green synthesis using plants is facile, safe, cost and time efficient, convenient way.

Furthermore, it is environment friendly, easily scaled up for large‐scale synthesis and in this method there is no need to use expensive tools and equipment, complicated procedures, high pressure, temperature, toxic chemicals and energy. The rich vegetation which covers Kerman province and easy access to plant sources are two most important factors in choosing the production of AgNPs using plant systems. Collectively, our finding revealed that the biosynthesis of AgNPs by using T. resupinatum seed exudates has an antifungal effect against the phytophatogenic fungi. In summary, this study provides information about the characterisation of the synthesised AgNPs using T. resupinatum seed exudates by UV–Vis spectroscopy, TEM, FTIR and XRD. Moreover, the antifungal activity of AgNPs in vitro condition was showed. The antifungal effect of AgNPs was size dependent and dose dependent. Further studies are recommended to be carried out on the efficacy of bio AgNPs against various pathogenic fungi. Also, animal models and clinical studies will need to be performed to get a better understanding of the antifungal efficiency of AgNPs.