Biosynthesised silver nanoparticles using aqueous leaf extract of Tagetes patula L. and evaluation of their antifungal activity against phytopathogenic fungi

Abstract

In the recent decades, nanotechnology is gaining tremendous impetus due to its capability of modulating metals into their nanosize, which drastically changes the chemical, physical, biological and optical properties of metals. In this study, silver nanoparticles (AgNPs) synthesis using aqueous leaf extracts of Tagetes patula L. which act as reducing agent as well as capping agent is reported. Synthesis of AgNPs was observed at different parameters like temperature, concentration of silver nitrate, leaf extract concentration and time of reduction. The AgNPs were characterized using UV‐vis spectroscopy, scanning electron microscope with energy dispersive spectroscopy, transmission electron microscopy with selected area electron diffraction, X‐ray diffraction, Fourier transform infrared and dynamic light scattering analysis. These analyses revealed the size of nanoparticles ranging from 15 to 30 nm as well revealed their spherical shape and cubic and hexagonal lattice structure. The lower zeta potential (−14.2mV) and the FTIR spectra indicate that the synthesized AgNPs are remarkably stable for a long period due to the capped biomolecules on the surface of nanoparticles. Furthermore, these AgNPs were found to be highly toxic against phytopathogenic fungi Colletotrichum chlorophyti by both in vitro and in vivo and might be a safer alternative to chemical fungicides.

1 Introduction

Noble metal nanoparticles are gaining lot of significance for the past few years due to their applicability in the field of medicine, agriculture, biology, material science, electronics, physics and chemistry [1]. Biosynthesis of metal nanoparticles using plant materials such as tissues, plant extracts and living plant has received considerable attention due to its environmentally benign nature. This method is free from the use of harsh, toxic and expensive chemicals and very cost effective, therefore it can be considered as an economic and valuable alternative for the large‐scale synthesis [2].

A number of approaches are available for the synthesis of silver nanoparticles (AgNPs) for example, reduction in solutions [3], chemical and photochemical reactions in reverse micelles [4], thermal decomposition of silver compounds [5], radiation assisted [6], sonochemical [7] microwave‐assisted process [8] and recently via green chemistry route [2, 9]. The use of environmentally benign materials like plant leaf extract [10], bacteria [11], fungi [12] and enzymes [13] for the synthesis of AgNPs offers numerous benefits of eco‐friendliness and compatibility for pharmaceutical and other biomedical applications as they do not use toxic chemicals for the synthesis protocol.

From ancient past, it has been identified that silver and its compounds are effective antimicrobial agents [14]. AgNPs are found to have anti‐bacterial [15] and anti‐fungal [16, 17] properties against phytopathogenic bacteria and fungi. This is due to their extremely large surface which provides better contact with microorganisms. The AgNPs get attached to the cell wall of microbes, which disturbs the permeability of cell wall and cellular respiration. The nanoparticles also cause cellular damage by reacting with phosphorus and sulphur containing compounds, such as DNA and protein as it can even penetrate deep inside the cell wall [18]. Therefore silver‐based compounds are now extensively used in many bactericidal applications [19].

In this study, we propose to use leaves of marigold (Tagetes patula L.) for synthesis of AgNPs. In marigold, once flowers are plucked from the plants, the remaining parts has been discarded hence, we report the beneficial use of these discarded plant leaves towards synthesising AgNPs. These nanoparticles were also checked for stability and antifungal activity against pathogenic fungi Colletotrichum chlorophyti both in vivo and in vitro.

2 Material and method

2.1 Plant material and preparation of the extract and synthesis of silver nanoparticles

Green leaves of marigold (T. patula L.) were used to make the aqueous extract and 2 mM aqueous solution of silver nitrate (AgNO₃) was prepared and used for the synthesis of AgNPs. Ten millilitres of leaves extract were added into 90 mL of aqueous solution of 2 mM AgNO₃ for reduction into Ag+ ions and kept at room temperature for 3 h. The effect of different concentrations of AgNO₃ was studied by carrying out the reaction at different concentration of AgNO₃, i.e. 0.5, 1, 2, 5 and 10 mM. The silver reduction was also studied at different temperatures, i.e. 25°–60°C. The AgNPs thus obtained after complete reduction was purified by repeated centrifugation at 10,000 rpm for 10 min followed by redispersion of the pellet in deionised water. The samples were stored at room temperature in dark.

2.2. Characterisation of AgNPs

2.2.1 UV–visible spectroscopy (UV–vis spectral) analysis

The reduction of pure Ag⁺ ions to AgNPs was monitored by measuring the UV–vis spectrum of the reaction medium from 30 min–5 h. UV–vis spectral analysis was done by using Nanophotometer (Implen, Germany) and absorbance was recorded at a wavelength range of 300–600 nm [20].

2.2.2 X‐ray diffraction (XRD) analysis

The AgNPs solution was repeatedly centrifuged and freeze dried. Thus the structure and composition of obtained purified AgNPs were analysed by an X'Pert Pro X‐ray diffractometer (PAN analytical BV, the Netherlands) operated at a voltage of 40 kV and a current of 30 mA with Cu Kα radiation in θ –2θ configurations. The crystallite domain size was calculated from the width of the XRD peaks, assuming that they are free from non‐uniform strains, using the Scherrer formula.

$$D=0.94\lambda /\beta \cos \theta$$

where D is the average crystallite domain size perpendicular to the reflecting planes, λ is the X‐ray wavelength, β is the full width at half maximum (FWHM) and θ is the diffraction angle. To eliminate additional instrumental broadening, the FWHM was corrected, using the FWHM from a large grained Si sample.

$$\beta \mathrm{corrected}={\left({\mathrm{FWHM}}_{\mathrm{sample}}^2-{\mathrm{FWHM}}_{\mathrm{si}}^2\right)}^{1/2}$$

This modified formula is valid only when the crystallite size is <100 nm [21].

2.2.3 Fourier Ttransform Iinfra‐Rred Sspectroscopy (FTIR) analysis

FTIR spectra of the samples were measured using FTIR Nicolet Avatar 660 (Nicolet, USA) in the region of 400–4000 cm⁻¹ for determine the functional groups which are responsible for the reduction and capping of AgNPs. The AgNPs was centrifuged and freeze dried (concentrator, Labtech) and obtained dried nanoparticles were analysed. The aqueous leaf extract were also analysed along with AgNPs.

2.2.4 Transmission electron microscopy (TEM) with selected area electron diffraction (SAED) analysis

Samples for TEM analysis were prepared on carbon‐coated copper TEM grid on Joel JEM‐1400 TEM machine. The films on the TEM grid were allowed to stand for 2 min following which the extra solution was removed using a blotting paper and the grid was allowed to dry under infrared lamp prior to measurement. The sharp spots in the SAED pattern indicated the crystalline nature of AgNPs.

2.2.5 Scanning electron microscope (SEM) with electron diffraction spectroscopy (EDS) analysis

Thin films of the sample were prepared on a carbon‐coated copper grid by just dropping a very small amount of the sample on the grid and then the film on the SEM grid were allowed to dry by putting it under a mercury lamp for 5 min. EDS measurements of the T. patula L. reduced AgNPs drop coated onto Si (111) wafers were performed on a Leica Stereoscan‐440 SEM instrument equipped with a Phoenix EDAX attachment.

2.2.6 Dynamic light scattering (DLS) and zeta potential analysis

Zeta potential and size distribution of AgNPs were determined by dynamic light scattering (DLS) using Malvern Zeta‐sizer Nanoseries (UK), measurements were carried out for sizes ranging from 0.1 nm to 10 µm.

2.2.7 TG/DTA studies

The reaction type and weight loss have been confirmed using thermogravimetric analysis (TGA)/derivative thermogram (DTG) thermal system (DTG‐60, Shimadzu, Japan).

2.2.8 In vitro antifungal assay

Poison food technique was used to measure antifungal activity. Four concentrations (25, 50, 75, 100) of AgNPs were used in antifungal test against three plant pathogenic fungi Colletotrichum chlorophyti. The inoculated plates were compared with control (without nanoparticles) to calculate the % inhibition rate of mycelia of the pathogen [22]:

$$\mathrm{Inhibition}\mathrm{rate}(\text{\%})=\frac{M_{\mathrm{c}}-M_{\mathrm{t}}}{M_{\mathrm{c}}}\times 100$$

where M _c is mycelial growth in control; M _t is mycelial growth in treatment.

2.2.9 In vivo antifungal assay

To determine the efficacy of AgNPs against Anthracnose (Colletotrichum chlorophyti) of Safed Musli (Chlorophytum borivilianum) in the field, an experiment was carried out and the field plants were infected using inoculums prepared in the laboratory. 100 ppm AgNPs were used along with other organic fungicides, namely 0.01% biodynamic mixture (BD501) and 3% neem oil. The AgNPs and other organic fungicide formulations were sprayed three times, first on the appearance of disease and second and third spraying was done at 15 days interval after first the spray. Observations were obtained after 15 days of the final treatment was given. Observations for the anthracnose disease were recorded on 0–5 disease rating scale to categorise plants into arbitrary classes based on disease progression. Per cent disease index (PDI) was calculated as

$$\mathrm{PDI}=\frac{\mathrm{Sum}\mathrm{of}\mathrm{all}\mathrm{individual}\mathrm{disease}\mathrm{ratings}}{\mathrm{Total}\mathrm{numbers}\mathrm{of}\mathrm{plants}\mathrm{assessed}\times \mathrm{Maximum}\mathrm{rating}}\times 100$$

While per cent efficacy of each treatment over control (PEDC) was calculated as

$$\mathrm{PDEC}=\frac{\mathrm{Infection}\mathrm{index}\mathrm{in}\mathrm{control}-\mathrm{Infection}\mathrm{index}\mathrm{in}\mathrm{treatment}}{\mathrm{Infection}\mathrm{index}\mathrm{in}\mathrm{control}}\times 100$$

2.2.10 Statistical analysis

The observations recorded for in vitro antifungal assay were subjected to statistical analysis such as standard deviation using standard equations. Furthermore, for determining significant difference among variable treatment on different AgNPs, the analysis has been done using JMP software version 11 (SAS, 2009) using Turkey–Kramer HSD test at p  = 0.05.

3 Result

AgNPs exhibit yellow to brown colour in aqueous solution due to surface plasmon resonance (SPR) which arise due to the collective oscillation of free conduction electrons induced by an interacting electromagnetic field [20]. As the T. patula L. Leaf extract was mixed in the aqueous solution of the silver ion complex, it started to change the colour from watery to brown due to reduction of silver to silver ion by electron donor metabolites present in the leaf extract, which is the first visible indication of AgNPs synthesis. Absorption spectra of AgNPs formed in the reaction media revealed an absorbance peak at 420 nm (Fig. 1). Broadening of the peak indicated that the particles are polydispersed. Analysis at different temperature 25–60°C showed the maximum synthesis of AgNPs occurs at temperature 40°C as shown in Fig. 2. Analysis of AgNPs synthesis at different concentration of AgNO₃ 0.5, 1, 2, 5 and 10 mM showed the synthesis of AgNPs increased with AgNO₃ concentration (Fig. 2). The synthesis of AgNPs occurs at different concentration of plant leaf extract like 1, 2.5, 5, 7.5 and 10 mL per 100 mL of AgNO₃ solution (Fig. 2). The result indicated that a very rapid and increased AgNPs synthesis with increased concentration of leaf extract. Temperature, pH, time and others are only some few examples of reaction conditions affecting the size and shape of AgNPs which can be observed through changes in SPR phenomena [23]. Based on the above experiment, we had optimised the AgNPs synthesis with 2 mM AgNO₃ along with 10 mL aqueous leaf extract at room temperature for 3 h.

Fig. 1.

UV–vis absorption spectra of AgNPs synthesised by using T. patula leaf extract

Fig. 2.

UV–vis absorption spectra of effect of different parameters on synthesis of AgNPs at different concentration of

(a) AgNO₃ concentration, (b) T. patula L. leaf extract concentration, (c) Temperature

Further, these AgNPs were characterised by XRD, FTIR, DLS and zeta potential, TEM with SEAD and SEM with EDS. The typical XRD pattern and TEM microgram revealed that the AgNPs is in mixed phase (cubic and hexagonal crystal lattice structures) of AgNPs ranged from 15 to 30 nm and majority of the AgNPs were scattered with only a few of them showing aggregation. XRD is used for the phase identification and characterisation of the crystal structure of the nanoparticles [24]. The result showed bands at angles of 38.11, 44.38, 64.44 and 76.57. This XRD result reveals silver crystal formation which, respectively, corresponds to 111, 200, 220 and 311 Bragg's planes. Thus it can be concluded that the silver crystallites are FCC structured. The unassigned band at 2θ  = 32.28° denoted by (*) in Fig. 3 is thought to be related to crystalline and amorphous organic phases.

Fig. 3.

XRD micrograph of AgNPs synthesised using T. patula L. leaf extract

FTIR absorption spectra of Ag ions are shown in Fig. 4 observed in the region of 400–4000 cm⁻¹ are 3170.97, 1593.20, 1388.75, 12611.45, 1072.42, 497.63 and 420.48 cm⁻¹. These absorbance bands are known to be associated with the stretching vibrations for –O–H [carboxylic acid], –C–C‐[(in‐ring) aromatic], –C(CH₃)₃, –C–N‐[aromatic amines], –C–N‐[aliphatic amines] and alkyl, groups, respectively. This strong absorption is due to the presence of surface adsorbed alcohols (carbohydrates etc.) and phenols (polyphenols). FTIR study indicates that the carboxyl (−C=O), hydroxyl (−OH), and amine (N–H) groups in leaf extract are mainly involved in the reduction of Ag+ to Ag nanoparticles. This was confirmed by the FTIR spectra of aqueous leaf extract which also shows the absorbance in similar regions (Data were provided in supplementary sheet.). The FTIR measurements were carried out to determine the biomolecules specifically bound on the silver particles that are involved in the reduction, capping and stabilisation [9]. The AgNPs were also found to be luminescent may be due to presence of biochemical/phenolics/antioxidants present in plant extract (data not shown).

Fig. 4.

FTIR micrograph of AgNPs synthesised using T. patula L. leaf extract

TEM analysis of the AgNPs synthesised using aqueous extract confirmed their monodispersity and narrow size distribution (Fig. 5). The majority of nanoparticles were spherical in shape and between 15 and 30 nm in size. The size and shape of AgNPs were remarkably stable for long time (1 year in dark, at ambient temperature) and remained fixed even after a year. The single crystalline nature of the biogenic AgNPs is reflected in the crystalline nature of the SAED spots (Fig. 5) corresponding to one of the structures of each nanoparticle. The SAED pattern of the nanoparticles showed cubic crystalline structure of silver indexed with different diffracting planes. The nanoparticles obtained were highly crystalline as shown by SAED pattern [25, 26].

Fig. 5.

TEM with SAED micrograph of AgNPs synthesised using T. patula L. leaf extract

The SEM image showing the high‐density AgNPs synthesised by the T. patula L. leaf extract further confirmed the development of silver nanostructures. Scanning electron microscopy is used for morphological characterisation at the nanometre to micrometre scale [27]. Elemental composition of metal nanoparticles is commonly established using energy dispersive spectroscopy (EDS) [28]. EDS measurements show the presence of metals gold, silica and silver as shown in Fig. 6 which shows that the AgNPs are pure in nature.

Fig. 6.

SEM with EDS micrograph of AgNPs synthesised using T. patula L. leaf extract

The DLS and zeta potential is used to characterise the surface charge, size distribution and potential stability of the nanoparticles suspended in a liquid. The solution contained particles of uniform sizes ranging from 10 to 50 nm. The size distribution of synthesised AgNPs was measured (Fig. 7). AgNPs were synthesised with a uniform distribution and an average particle size is −15 nm and similar results were also recorded by another method, CPS disc centrifuge, the Netherlands (Supplementary data sheet). The zeta potential graph of AgNPs resulted that the particles carry a charge of −14.2 mV upto 1 year and were stable at room temperature. The zeta potential was maintained at −14.2 mV even after 1 year of synthesis, hence we can say that this AgNps were stable for 1 year or more.

Fig. 7.

DLS and zeta potential analysis micrograph of AgNPs synthesised using T. patula L. leaf extract

Thermal properties TGA and DTG spectra have been recorded in temperature range from room temperature to 900°C. TGA and DTG curves of dried powder of AgNPs and dried leaf powder were performed (Fig. 8). It is observed from TGA curve that dominant weight loss of the sample occurred in temperature region between 200 and 300°C. There is not much weight loss <200°C and >300°C. In dried leaf samples, the initial weight loss from temperature 25°C to 115°C was attributed as evaporation of moisture content, which was about 10 wt%. Extensive weight loss in biomass after 200°C was observed due to the release of volatile compounds in leaf extract. After this final degradation temperature, there was gradual decrease in weight loss. This was attributed to the burning of remaining solid residue or char, which progressed until 800°C (Supplementary data sheet). The pattern of weight loss is similar in AgNPs and leaf powder of T. patula indicates the evaporation of water and the volatile organic components involved in capping and stabilisation of AgNPs. DTG plot displays an intense exothermic peak between 200 and 300°C which mainly attributed to crystallisation of AgNPs [29].

Fig. 8.

Thermal properties TGA and DTA spectra of AgNPs synthesised using T. patula L. leaf extract

Furthermore, these AgNPs were observed highly toxic against selected phytopathogen Colletotrichum chlorophyti. Antifungal activity of AgNPs was tested by Poison Food technique using PDA media (Fig. 9). AgNPs show significant antifungal activity against these phytopathogenic fungi Colletotrichum chlorophyti (Table 1). Table 2 summarised the results AgNPs and other organic fungicidal formulations against Anthracnose (Colletotrichum chlorophyti) of Safed Musli (Chlorophytum borivilianum Sant. and Fernand) under inoculation conditions. Under field condition mean data revealed that maximum per cent efficacy of disease control 82.34 per cent were observed in foliar sprays of AgNPs followed by BD‐501. In this treatment 6.50 mean PDI was also observed when compared with 36.8 mean PDI in the control. The AgNPs at the concentration of 100 ppm were found to be best for controlling anthracnose at field conditions. In previous studies, antifugal effect of AgNPs reported against Aspergillus, Candida and Saccharomyces [30], Trichosporon asahii [31] and anti‐bacterial effect against soil bacterium [32].

Fig. 9.

A representative photograph of antifungal bioassay of AgNPs against Colletotrichum chlorophyti mycelia growth inhibition by poison food technique

(a) Control, (b) 25 ppm, (c) 50 ppm, (d) 75 ppm and (e) 100 ppm

Table 1.

Antifungal activities of AgNPs on in vitro mycelia growth of Colletotrichum chlorophyti

Treatment	% inhibition mycelia growtha
controlb	0.0 D
25 ppm	24.88 ± 0.85C
50 ppm	38.16 ± 0.76B
75 ppm	52.33 ± 1.52B
100 ppm	63.01 ± 0.97A

^a Each value is mean of three replicates. Mean ± SE followed by same letter in column of each treatment is not significant difference at p  = 0.05 by Tukey–Kramer HSD test, % inhibition rate was calculated compared with the germination of the control (0%).

^b Control without any formulation.

Table 2.

Evaluation of organic fungicidal formulations against Anthracnose (Colletotrichum chlorophyti) of Safed Musli (Chlorophytum borivilianum Sant. and Fernand) under inoculation condition

S. no.	Spray treatmenta	PDI over inoculated control	PEDC over inoculated control
1.	BD‐501 (0.01%)	10.25 (18.67)	72.15 (58.15)
2.	Neem oil (3.0%)	14.21 (22.15)	61.39 (51.58)
3.	Silver nanoformulation (0.01%)	6.50 (15.77)	82.34 (65.15)
4.	Inoculated untreated control	36.8 (37.35)	0.00 (0.00)
SEM±		0.47	1.74
CD 5%		1.28	4.42
CD 1%		1.90	6.92
CV %		3.25	3.66

PDI, per cent disease incidence; PEDC, percentage efficacy of disease control; CD, confidence distribution; CV, coefficient of variance.

^a Figures in parentheses are angular transformed values.

Earlier studies suggest that these AgNPs are found to have anti‐bacterial [33], antifungal [16] and antiviral [34] properties against phytopathogens. The probable mechanism of antimicrobial of AgNPs is interactions of these particles with phosphorus and sulphur containing compounds, such as DNA and protein, which prevent the ability of DNA to replicate since these particles can easily penetrate and mechanistically, the treatment of nanoparticles with fungal cells leads to rupture of cell wall as demonstrated by scanning electron microscope [18, 35]. The AgNPs synthesised via green route are highly toxic to phytopathogens and could be utilised as alternative source of bactericides and fungicides for the management of disease.

4 Conclusion

One of the first approach of using plants as a source for synthesising the metallic nanoparticles was alfalfa sprouts [36] which reported the formation of AgNPs using a living plant system. Plant extracts as reducing and capping agent could be considered attractive for nanotechnology [37]. Therefore, the use of plant extracts [38] in green synthesis has spurred numerous investigations and studies up till now. In this study, a rapid and eco‐friendly method for synthesis of AgNPs using aqueous leaf extracts of T. patula L. was demonstrated. The AgNPs were characterised by UV–vis spectroscopy, SEM with EDS, TEM with SAED, XRD, FTIR and DLS analysis. The metallic AgNPs were highly stable and crystalline in nature, which was confirmed by XRD and SAED pattern. The obtained results of the antifungal activity clearly reveal that synthesised AgNPs had shown best antimicrobial efficacy on selected phytopathogenic fungi Colletotrichum chlorophyti. On the basis of the comparison of these AgNPs with commercial organic fungicides, they could be pronounced to have even better antifungal activity. Applications of nanoparticles are emerging in crop protection and agriculture [39]. Biogenic synthesis of AgNPs is useful because of that they are economical, energy efficient and cost effective and provide healthier work places and communities, protecting human health and environment leading to lesser waste and safer products [38], they can be used to produce large quantities of nanoparticles that are free of contamination and have a well‐defined size and morphology [40]. The present study has opened up the possible way for synthesising antimicrobial AgNPs using natural biomolecules.