Comparative study of antifungal effect of green and chemically synthesised silver nanoparticles in combination with carbendazim, mancozeb, and thiram

Abstract

This study reports synthesis and characterisation of silver nanoparticles and their effect on antifungal efficacy of common agricultural fungicides. Silver nanoparticles were synthesised using biological and chemical reduction methods employing Elettaria cardamomum leaf extract and sodium citrate, respectively. Nanoparticles were then characterised using UV–Visible spectroscopy, X‐ray diffraction (XRD), transmission electron microscopy, and dynamic light scattering (DLS). While XRD assigned particles size of 31.86 nm for green and 41.91 nm for chemical silver nanoparticles with the help of the Debye–Scherrer formula, DLS specified monodisperse nature of both suspensions. Nanoparticles were tested individually and in combination with fungicides (carbendazim, mancozeb, and thiram) against fungal phytopathogens. Silver nanoparticles exhibited good antifungal activity and minimum inhibitory concentration (MIC) was observed in the range of 8–64 µg/ml. Also, they positively influenced the efficacy of fungicides. The mean MIC value (mean ± SD) for combination of all three fungicides with green AgNPs was 1.37 ± 0.6 µg/ml and for chemical AgNPs was 1.73 ± 1.0 µg/ml. Hence, it could be concluded that green AgNPs performed better than chemical AgNPs. Synergy was observed between green AgNPs and fungicides against Fusarium oxysporum. In conclusion, this study reports synthesis of monodisperse silver nanoparticles which serve as efficient antifungal agents and also enhance the fungicidal action of reported agricultural fungicides in combination studies.

1 Introduction

Fungal phytopathogens are the major cause of extensive yield and economic losses [1]. Fungal infections include active penetration and colonisation of plant parts by fungi affecting crop plants in all stages of life cycle from germination to fruiting [2]. They are also known to cause severe damage to fruits and vegetables post‐harvest [3]. For developing countries, spoilage of food crops could be even more detrimental as they are already suffering in terms of food security for growing populations [4]. News agencies have reported loss of millions of tons of food crops, fruits, and vegetables during transportation and storage, accounting for billions of rupees [5, 6]. The primary reason for post‐harvest spoilage of fruits and vegetables is their high moisture and nutrient content and can account for 5–50% loss of yield [7]. Additionally, low pH of fruits and post‐harvest decrease in infection resistance also contribute to increased decay [8]. Fungal species belonging to genera Alternaria, Aspergillus, Botrytis, Fusarium, Geotrichum, Gloeosporium, Monilinia, Mucor, Penicillium, and Rhizopus are most often associated with post‐harvest damage [9]. To combat these infections, chemical fungicides are the method of choice. Synthetic chemical fungicides are being routinely used for control of fungal infections on‐field as well as on crop produce, post‐harvest. Considering possible harmful effects of introducing chemicals in food supplies and their toxicological effects, many fungicides are found unsuitable for use [10]. Chemical residues of fungicides find their way in food, water, soil, and air which may lead to biomagnification in food chain. Adding to the grievousness of the situation, the intensive use of fungicides has led to emergence of fungicide‐resistance in fungal pathogens [11, 12, 13]. All this contribute to an urgent requirement for disease management strategies that minimise the use of harmful and toxic chemicals and offer eco‐friendly solutions with efficient pathogen control in crops and crop produce.

Nanotechnology practices are being implemented in almost all aspects of our daily lives [14, 15, 16, 17, 18, 19]. Silver nanoparticles (AgNPs) are particularly explored for their excellent antimicrobial potential [20, 21, 22, 23]. Silver has been known to possess antimicrobial properties and ability to fight against infections [24]. The inhibitory action of nanosilver shows a correlation with the shape and size of nanoparticles. It was reported that nanoparticles <10 nm in size were extremely effective against bacteria and even prevented the binding of viral particles to host cells [25, 26, 27]. The present work reports chemical synthesis of AgNPs with their application as potential antifungal agents. Green AgNPs were synthesised using leaf extract of Elettaria cardamomum. Leaves are the major source of photosynthetic products in plants. Further, carbohydrates such as glucose and starch provide increased stability to resultant suspensions [28]. Green and chemically synthesised AgNPs were then used in combination with fungicides registered for agricultural use in India, viz., carbendazim (methyl 2‐benzimidazole carbamate), mancozeb [manganese zinc ethylene bis (dithiocarbamate)], and thiram (bis (dimethyl thiocarbamoyl) disulphide) [29]. This is, to the best of our knowledge, the first report concerning combination study of silver nanoparticles with stated fungicides and depicts enhanced activity of fungicides in combination analysis.

2 Materials and methods

2.1 Materials

All the chemicals and media used in the present study were of A.R grade and were obtained from Sigma‐Aldrich Chemicals Pvt Ltd (Bangalore) and Hi‐Media Laboratories Pvt Ltd (Mumbai). Fungicides used were of analytical standard grade and were procured from Sigma‐Aldrich.

2.2 Synthesis of AgNPs

Green synthesis of AgNPs was performed using leaf extract of E. cardamomum at optimum conditions as described previously [30] and chemical AgNPs were synthesised by citrate reduction method as described by Van Dong et al. [31]. Briefly, 50 ml of 1 mM AgNO₃ was heated to bring to boil. While boiling and still heating, 5 ml of 1% trisodium citrate was added drop wise under continuous stirring using a magnetic stirrer. Heating and stirring was stopped when the colour of the solution changed from off‐white to greenish yellow. The solution was left undisturbed and allowed to cool. AgNPs obtained were purified by centrifuging at 15,000 rpm for 15 min, washing with ethanol twice and were lastly, re‐suspended in sterile de‐ionised water.

2.3 Characterisation of AgNPs

Purified AgNPs were characterised using UV–Visible spectroscopy, X‐ray diffraction (XRD), dynamic light scattering (DLS), and transmission electron microscopy (TEM). UV–Vis spectrum for AgNP suspension was noted in the range of 200–700 nm, using UV–Vis Spectrophotometer UV‐3092 from Labindia Analytical Instruments Pvt Ltd. For XRD analysis, AgNPs were concentrated by centrifugation and dried at 45°C on clean glass surface. The sample was powdered and XRD scan was performed with X‐ray wavelength of 1.5406 Å using Ultima IV (Rigaku, Japan). The 2θ /θ continuous scanning mode was used in the 2θ range of 20–80°. Hydrodynamic diameter and polydispersity index were noted as a function of time using DLS, which was performed using Zetasizer Nano (DLS, Malvern Instruments, Worcestershire, UK). The system employs a He/Ne red laser with a wavelength of 633 nm with the detector angle of 173°. For TEM analysis, AgNP suspension was sonicated for 15 min, coated onto a copper grid, and visualised using Morgagni 268D, FEI Electron Optics (USA), with the accelerating voltage of 200 kV. Images were viewed and analysed using Olympus siViewer and ImageJ softwares.

2.4 Antifungal activity

Synthesised AgNPs and test fungicides, namely carbendazim, mancozeb, and thiram, were tested for their antifungal potential against fungal phytopathogens, namely Alternaria alternata, Aspergillus niger, Botrytis cinerea, Fusarium oxysporum, and Penicillium expansum. Fungal isolates were maintained on potato dextrose agar and minimum inhibitory concentration (MIC) analysis was performed using potato dextrose broth.

Preliminary studies for testing antifungal potential of silver nanoparticles were performed using agar well diffusion assay as stated by Krishnaraj et al. [32] with minor modifications. One hundred microlitres of liquid spore suspension were evenly spread plated onto fresh PDA plates and two wells, 7 mm in diameter, were punched with a sterile borer. Fifty microlitres of nanoparticle suspension were added as test solution (T well) and 10 µl of 2% ketoconazole was used as positive control (PC well). The plates were then incubated in the upright position for 2–3 days at 28°C. To prepare inoculum for MIC analysis, conidia of the stated cultures were collected from 5‐day‐old fungal lawns and diluted to adjust spore count to 1 × 10⁵ c.f.u./ml. MIC analysis was performed using broth dilution assay and results were noted based on visual examination of tubes with 2× serially diluted nanoparticles. Tubes were observed for complete (100%) inhibition of growth and absence of any fungal fibre, for all the experiments. The initial concentration of AgNPs and fungicides used was 256 µg/ml and 100 µl of diluted spore suspension was added to 2 ml of reaction volume. Results were noted after 3‐day incubation at 28°C [33]. For combination studies, AgNPs and fungicides were mixed in equal concentration, incubated overnight, and tested to ascertain the MIC value in a manner similar to that stated above [34]. Six combinations resulted viz. carbendazim with green AgNPs (CGA) and chemical AgNPs (CCA), mancozeb with green AgNPs (MGA) and chemical AgNPs (MCA), and thiram with green AgNPs (TGA) and chemical AgNPs (TCA). Sterilised broth with no fungal inoculation was used as blank for the study. One millilitre broth inoculated with 100 µl of spore suspension, in the presence and absence of 1 ml 2% ketoconazole, were used as positive and negative controls, respectively. All tubes corresponding to test samples, blank and control, were incubated in the same conditions.

2.5 Statistical analysis

A paired sample t ‐test was applied to combination tests to ascertain if there was a statistically significant mean difference between the MIC values observed for fungicides alone and fungicide–nanoparticle combinations. Origin Pro 8.0 was used for carrying out statistical studies.

2.6 Determination of fractional inhibitory concentration

For determining fractional inhibitory concentration (FIC) indices for combinations tested, a two‐dimensional chequerboard analysis was performed. Two‐fold dilutions of AgNPs (ranging from 128 to 0.25 µg/ml) and fungicides (ranging from 16 to 0.0625 µg/ml) were used in 96‐well microtitration plates. Fifty microlitres of all dilutions of AgNPs were mixed individually with 50 µl of all dilutions of fungicides, as described for combination studies. These were then inoculated with 100 µl of count adjusted spore suspension and incubated at 28°C for 3 days. For all wells showing no visible fungal growth, FIC values were calculated as: FIC_AgNP  = MIC_AgNP in combination/MIC_AgNP alone, FIC_Fungicide  = MIC_Fungicide in combination/MIC_Fungicide alone, and FICI = FIC_AgNP  + FIC_Fungicide [35, 36]. Results obtained were interpreted as ‘synergy’ (FICI ≤ 0.5), ‘no interaction’ (FICI > 0.5–4.0), and ‘antagonism’ (FICI > 4.0) [37].

3 Results and discussion

3.1 Synthesis and characterisation of AgNPs

Green synthesis of AgNPs was performed using E. cardamomum leaf extract and UV–Vis spectroscopy resulted in the absorption peak at 408 nm (Fig. 1 a). Chemical AgNPs were synthesised using sodium citrate as the reducing agent. The addition of sodium citrate to boiling silver nitrate parent solution resulted in colour change from transparent to greenish yellow and UV–Vis analysis showed an intense peak at 433 nm (Fig. 1 b). The proposed mechanism for reaction is as follows (1):

$$4\mathrm{A}{\mathrm{g}}^{+}+{\mathrm{C}}_6{\mathrm{H}}_5{\mathrm{O}}_7\mathrm{N}{\mathrm{a}}_3+2{\mathrm{H}}_2\mathrm{O}\to 4\mathrm{A}{\mathrm{g}}^0+{\mathrm{C}}_6{\mathrm{H}}_5{\mathrm{O}}_7{\mathrm{H}}_3+3\mathrm{N}{\mathrm{a}}^{+}+{\mathrm{H}}^{+}+{\mathrm{O}}_2\uparrow .$$ (1)

Fig. 1.

UV–Visible spectral plots for

(a) Green AgNPs, (b) Chemical AgNPs

Colloidal silver generated in the process maintains a net negative charge on the surface due to an adsorbed layer of citrate. This stabilising layer is responsible for electrostatic repulsion among the particles, thereby, contributing to the formation of monodisperse, non‐aggregating nanoparticle suspension [31]. Hence, sodium citrate acts as both reducing and stabilising agent.

XRD analysis of synthesised AgNPs was performed to establish their crystalline nature (Fig. 2). XRD pattern observed in the present study showed intense peaks at 2θ values of 38.20°, 44.24°, 64.48°, and 77.48° for green AgNPs and 38.08°, 44.30°, 64.44°, and 77.38° for chemical AgNPs. These peaks were in accordance with those reported in the JCPDS file for crystalline silver: 04‐0783. Data analysis and peak indexing concluded that the stated 2θ values correspond to (111), (200), (220), and (311) planes of Bragg's reflections for FCC structures. Bragg's law equation (2) was employed for the calculation of interplanar d ‐spacing (Table 1) and the Debye–Scherrer formula (3) was used for particle size (D) calculation [38]

$$2d\sin \theta =n\lambda .$$ (2)

$$D=0.9\lambda /\beta \cos \theta ,$$ (3)

where n  = 1, θ is the angle of diffraction, β the full width at half maximum (calculated using OriginPro 8.0), and λ the wavelength of X rays used (0.154 nm). The intense peak corresponding to the plane (111) was used for calculating particle size. The average particle size, as calculated using the Debye–Scherrer formula, was found to be 31.86 nm for green AgNPs and for chemical AgNPs was 41.91 nm.

Fig. 2.

X‐ray diffractograms for

(a) Green AgNPs, (b) Chemical AgNPs

Table 1.

XRD peak indexing and d ‐spacing for green and chemical AgNPs

Peak 2θ	θ	sin θ	d  = nλ /2 sinθ, Å	d, nm	hkl
Green AgNPs
38.20	19.10	0.327	2.3765	0.23765	111
44.24	22.12	0.376	2.0370	0.20370	200
64.48	32.24	0.533	1.4398	0.14398	220
77.48	38.74	0.626	1.2300	0.12300	311
Chemical AgNPs
38.08	19.04	0.326	2.3629	0.23629	111
44.30	22.15	0.377	2.0432	0.20432	200
64.44	32.22	0.533	1.4452	0.14452	220
77.38	38.69	0.625	1.232	0.1232	311

As seen from the UV–Vis spectral plots of green and chemical AgNPs, the peak for the latter depicts a prominent red shift towards larger wavelength. Wavelength shifts are associated with the size of nanoparticles, and this is further confirmed by XRD analysis, which shows larger particle size for chemical AgNPs.

TEM micrograph showed nanosized particles for both green and chemical AgNP suspensions with size <100 nm (Fig. 3). DLS plot depicts that most of the green synthesised AgNPs are clustered in the region of 9–100 nm, whereas for chemical AgNPs, size distribution depicted two peaks in the regions corresponding to 6–20 and 20–110 nm (Fig. 4). DLS gave an average hydrodynamic diameter of 32.39 and 45.89 nm for green and chemical AgNPs, respectively. This value is larger than that calculated from XRD data and is attributed to the presence of capping layer on the surface of the nanoparticles [39]. The polydispersity index value (<0.4) specifies monodisperse nature of AgNP suspension indicating a stable colloid and depicts minor aggregation, if any.

Fig. 3.

TEM micrographs and particle size distribution for

(a) Green AgNPs, (b) Chemical AgNPs

Fig. 4.

DLS plots for

(a) Green AgNPs, (b) Chemical AgNPs

3.2 Antifungal potential of AgNPs

Agar well diffusion was used for the preliminary study of inhibition of fungal growth by nanoparticles. Varying diameters for zone of inhibition were observed for all the test fungi. Since this was performed for initial screening of antifungal activity, analysis was confined to only presence and absence of inhibition zones. Fig. 5 depicts inhibition of A. alternata and P. expansum by chemical and green synthesised AgNPs, respectively. Well‐marked ‘T’ depicts zone of inhibition by AgNPs, while well‐marked ‘PC’ depicts zone of inhibition observed for ketoconazole. MIC analysis was then performed, based primarily on visual inspection for the presence of fungal growth in increasing dilutions of nanoparticles. Fungal growth failed to occur at high concentration of nanoparticles and increased steadily at lower concentrations. Results were noted as the presence or absence of growth at different dilutions. MIC values for green AgNPs were the same as reported previously [30]. Chemical AgNPs were also found to be effective against all test fungi (Table 2).

Fig. 5.

Inhibition of A. alternata and P. expansum by AgNPs

Table 2.

MICs of synthesised nanoparticles

Fungus	MIC, µg/ml
	Green AgNPs	Chemical AgNPs
A. alternata	32	64
A. niger	8	8
B. cinerea	32	32
F. oxysporum	32	64
P. expansum	64	64

Lowest MIC was recorded for A. niger as 8 µg/ml and highest MIC value was recorded as 64 µg/ml for A. alternata, F. oxysporum, and P. expansum. MIC values for A. alternata and F. oxysporum were found to increase from 32 to 64 µg/ml when compared with that observed with green AgNPs. Since green AgNPs were synthesised using Elettaria leaf extract, which has been shown to possess little or no antimicrobial activity [40], it could be postulated that size difference in both classes of AgNPs is primarily responsible for better antifungal potential of biosynthesised green AgNPs. A range of probable MIC values have been observed for AgNPs by various researchers and values as low as 0.5–1 µg/ml could be seen in the literature for various fungal isolates [41]. Shanmugaiah et al. [42] synthesised AgNPs using bacterial isolate Streptomyces sp.VSMGT1014 and reported excellent antibacterial and antifungal activity. They have reported MIC values of 40 µg/ml for A. alternata and 50 µg/ml for Fusarium udum. Further, a very similar MIC value of 32.1 µg/ml for F. oxysporum was reported for AgNPs synthesised using bulb extract of Allium cepa [43].

When AgNPs were used in combination with fungicides, they were found to positively influence the inhibitory action of all the fungicides and MIC values were found to decrease by at least one dilution (Tables 3, 4, 5). Working with carbendazim and thiram gave less pronounced effects, but MGA gave a two dilution decrease in MIC values. F. oxysporum was found to be the most sensitive fungus and showed increased inhibition at lower dose of 0.5 µg/ml for CGA compared to 2 µg/ml for carbendazim alone and 1 µg/ml for MGA compared to 4 µg/ml for mancozeb alone. P. expansum was the least sensitive for all, but one (CGA), tested combinations where only one dilution decrease in MIC was observed. When comparing all the three fungicides, mancozeb was the most effective fungicide in combination studies. Thiram was found to be the least effective in combination studies with both TGA and TCA giving only one fold dilution difference for all the fungi when compared with inhibition by thiram alone. The paired sample t ‐test analysis was performed to analyse the significance of difference in means of the combination samples compared with individual fungicides. The t ‐ statistic values ranged between 3.09 and 6.53 which was higher than the p ‐value of 2.78 at significance level of 0.05 and degree of freedom = 4, implying statistical significance of the results.

Table 3.

Statistical comparison of MIC values obtained for carbendazim alone and in combination with AgNPs

Fungus	Carbendazim	CGA	CCA
A. alternata	2	1	1
A. niger	1	0.5	0.5
B. cinerea	1	1	1
F. oxysporum	2	0.5	0.5
P. expansum	2	0.5	1
mean	1.6	0.7	0.8
SD	0.55	0.27	0.27
SEM	0.25	0.12	0.12
t value	—	3.09	3.14
inference	—	significant	significant

Table 4.

Statistical comparison of MIC values obtained for mancozeb alone and in combination with AgNPs

Fungus	Mancozeb	MGA	MCA
A. alternata	4	2	2
A. niger	8	2	4
B. cinerea	8	2	4
F. oxysporum	4	1	2
P. expansum	4	2	2
mean	5.6	1.8	2.8
SD	2.19	0.45	1.10
SEM	0.98	0.20	0.49
t value	—	4.15	5.72
inference	—	significant	significant

Table 5.

Statistical comparison of MIC values obtained for thiram alone and in combination with AgNPs

Fungus	Thiram	TGA	TCA
A. alternata	2	1	1
A. niger	4	2	2
B. cinerea	4	2	2
F. oxysporum	2	1	1
P. expansum	4	2	2
mean	3.2	1.6	1.6
Sd	1.10	0.55	0.55
SEM	0.49	0.25	0.25
t value	—	6.53	6.53
inference	—	significant	significant

Green AgNPs performed marginally better than chemical AgNPs in the above‐stated studies. The only two dilution decrease was observed for F. oxysporum for CCA (0.5 µg/ml) when compared with carbendazim alone (2 µg/ml); in all other experiments, we obtained only one dilution difference in the MIC values. Better performance of green AgNPs could be attributed to small average size [44] as well as the presence of NPs towards a smaller size range than chemical AgNPs. AgNPs play a major role in disruption of cell wall and plasma membrane of target microorganisms, thereby, leading to cavity formation and electrolyte imbalance [45, 46]. The ruptured membrane allows for better penetration of fungicides inside the cells and combined effects of silver ions on DNA, RNA, and proteins [47, 48] and fungicides led to improved inhibition of fungal targets.

Chequerboard analysis was performed for determination of FIC indices for all tested combinations; the results of which are summarised in Tables 6 and 7. While all the combinations showed improved antifungal activity, synergistic interactions were observed for A. niger (MGA combination) and F. oxysporum (CGA, MGA, TGA, MCA, and TCA combinations). For other fungi and tested combinations, FICI values ranged from 0.625 to 1.0 emphasising ‘no interaction’ between AgNPs and fungicides in the present study [37].

Table 6.

FIC indices for combination of green AgNPs with fungicides

Fungus	FICI (µg/ml)a
	CGA	MGA	TGA
A. alternata	0.75	0.625	0.75
A. niger	0.625	0.5	0.75
B. cinerea	1.0	0.75	0.75
F. oxysporum	0.5	0.5	0.5
P. expansum	0.75	0.75	0.75

^a FICI = (MIC_GreenAgNP in combination/ MIC_GreenAgNP alone) + (MIC_Fungicide in combination/MIC_Fungicide alone).

Table 7.

FIC indices for combination of chemical AgNPs with fungicides

Fungus	FICI (µg/ml)a
	CCA	MCA	TCA
A. alternata	0.75	0.75	1.0
A. niger	1.0	0.75	0.75
B. cinerea	1.0	0.75	1.0
F. oxysporum	0.625	0.5	0.5
P. expansum	0.75	1.0	1.0

^a FICI = (MIC_ChemicalAgNP in combination/MIC_ChemicalAgNP alone) + (MIC_Fungicide in combination/MIC_Fungicide alone).

The results obtained, herewith, were in accordance with those already reported by various authors. AgNPs have been investigated for their antimicrobial activity individually and in combination with various antibiotics and antifungal drugs. AgNPs were investigated for their synergism with fluconazole and tested against various fungal pathogens. A significant increase in the inhibitory zones of inhibition for Candida albicans, Trichoderma sp. and Phoma glomerata was observed, with C. albicans being the most sensitive to the combination [49]. Panáček et al. [50] performed microdilution assay for determination of MIC with geometric dilutions of antibiotics, to which AgNPs were added at individual MIC of AgNPs against the tested bacteria. They reported strong synergistic effects of AgNPs, in combination with an array of antibiotics against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Such combinations have also been found to be effective against multidrug‐resistant bacteria. Hence, combination studies emphasise excellent antimicrobial potential of AgNPs when used in conjunction with already existing antimicrobial agents.

4 Conclusion

The present study reports comparative analysis of the effect of green and chemical AgNPs on the efficacy of commercial agricultural fungicides. Green AgNPs were synthesised using E. cardamomum leaf extract and chemical reduction was performed using sodium citrate. Characterisation was performed using UV–Vis spectroscopy, XRD, TEM, and DLS. Antifungal efficacy of green and chemical AgNPs was examined and MIC values in the range of 8–64 µg/ml were observed for tested fungal phytopathogens. When AgNPs were used in combination with fungicides, positive influence on inhibitory action of all the fungicides was observed. F. oxysporum was found to be the most sensitive fungus and P. expansum was the least sensitive for almost all tested combinations. Among fungicides, mancozeb was most effective, while thiram was found to be the least effective in combination studies with both green and chemical AgNPs. Synergistic interaction was observed between green AgNPs and fungicides, carbendazim, mancozeb, and thiram, when used in combination against F. oxysporum. Conclusively, we report effective combinations with excellent antifungal potential which can be further developed into commercial formulations for agricultural use.