Antifungal effect of green synthesised silver ...

IET Nanobiotechnology Research Article

Antifungal effect of green synthesised silver nanoparticles against Setosphaeria turcica

ISSN 1751-8741 Received on 30th September 2016 Revised 7th February 2017 Accepted on 18th May 2017 E-First on 27th July 2017 doi: 10.1049/iet-nbt.2016.0200 www.ietdl.org

Weidong Huang1, Yu Bao1, Haiming Duan1, Yaling Bi1, Haibing Yu1 1College

of Agriculture, Anhui Science and Technology University, Fengyang 233100, People's Republic of China E-mail: [email protected]

Abstract: Green synthesis of silver nanoparticles (AgNPs) is an interesting issue of the nanoscience and nanotechnology due to their unique properties. In the present study, Ginkgo biloba L. leaf extract was used to synthesise AgNPs. The effects of quantity of leaves, concentration of Ag nitrate (AgNO3), reaction temperature, and pH were studied to discover the optimal synthesis system. In addition, antifungal effect of AgNPs against Setosphaeria turcica was measured through inhibition zone method. The optimal biosynthesis system contained 15 g of leaf, 8 mM AgNO3, and 80°C at pH 9.0. Under mentioned conditions, the resulting synthesised NPs were nearly spherical, with an average size of 14 nm. In tests, AgNPs synthesised at different pH resulted in different inhibition zones, diameters increased gradually at pH from 3.0 to 11.0, while antifungal effect reached maximum at 9.0. Results of this study offer a new approach for biological control plant pathogenic fungi, and it has potential application for screening novel fungistats with high efficiency and low toxicity.

1 Introduction Nanomaterials or nanoparticles (NPs) are defined as the size of which are in the range of 1–100 nm at least in one dimension [1]. Owing to the high surface area to volume ratio, NPs possess unique properties that differ from their bulk counterparts including optical, electrical, magnetic, chemical, and physical features [2]. Extensive application of nanomaterials benefits from the rapid development of nanoscience and nanotechnology. Metal NPs, especially for silver NPs (AgNPs) attract more and more researchers’ attention belonged to different disciplines. Synthesis of AgNPs can be implemented through multiple approaches such as physical [3, 4], chemical [5, 6], and biological methods [7, 8]. It has been found that there are several disadvantages for the former two methods such as energy consumption, usage of toxic chemicals, poor stability, and so on. Under the new situation of advocating green chemistry, biosynthesis method is increasingly valued. The materials used for biosynthesis of AgNPs contain bacteria [9, 10], actinomycetes [11], fungi [12, 13], and plants [8, 14]. Among these materials, plants exhibit more advantages such as less time, higher purity, better stability, and large-scale production. In recent years, more and more plants were used to synthesise AgNPs including Brassica rapa L. [15], Hibiscus rosa-sinensis [16], and Aristolochia indica L. [8]. It is well known that agricultural production is affected by various plant diseases to a large extent, leading to serious economic losses. In addition, unreasonable use of pesticides for a long time induced appearance of drug-resistant pathogens. In view of this, it is necessary to explore novel agents to replace existing pesticides in order to resolve the problem. AgNPs were proved to be of high antimicrobial activity during the past years including Staphylococcus aureus [17], Escherichia coli [18], Candida albicans [19], powdery mildews [20], HIV-1 [21, 22] etc. Although there were many researches about the interaction of AgNPs and microorganisms, the exact mechanism of which has not been totally understood. Up to now, there existed several possible explanations. (i) The extremely large surface area of AgNPs facilitates which contact with microorganisms [23]; (ii) direct interaction with cell membrane and damage it [24]; (iii) production of reactive oxygen species (ROS) after reacting with thiol (–SH) groups [25]; and (iv) release of Ag ions (Ag+) that inhibit respiratory enzyme and generate ROS [26]. IET Nanobiotechnol., 2017, Vol. 11 Iss. 7, pp. 803-808 © The Institution of Engineering and Technology 2017

In this research, the leaf extracts of Ginkgo biloba L. were used for rapid and simple biosynthesis of AgNPs, and the optimum biosynthesis process was achieved through adjusting quantity of leaves, concentration of Ag nitrate (AgNO3), pH, and reaction temperature. The biosynthetic AgNPs were characterised using ultraviolet–visible (UV–vis) spectrophotometer, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy dispersive X-ray (EDX). In addition, the antifungal activity of AgNPs was measured by inhibition zone method.

2 Materials and methods 2.1 Microorganism isolates The fungal used here was Setosphaeria turcica isolated from infected maize leaves in Fengyang, Anhui province, China. The single-spore isolate was preserved in the laboratory of Plant Protection at Anhui Science and Technology University. 2.2 Preparation of plant extract The extract of G. biloba L. was prepared as follows: leaves were thoroughly washed in sterile water and cut into small pieces, followed by drying on a clean bench. Pre-selected quantities of leaves (1, 2, 5, 10, 15, and 20 g) were added to 100 ml of deionised water separately, then heated at 90°C for 30 min. During the heating process, it is necessary to stir intermittently to heat uniformly. After that, the extract was filtered through filter paper and preserved for further experiments. 2.3 Optimal synthesis of AgNPs Biosynthesis of AgNPs was achieved through adding the filtrate to deionised water at the ratio of 1:9 (v:v), followed by reaction with AgNO3 under different temperatures. Colour change of solution indicated formation of AgNPs. The optimisation process was performed including different quantities of G. biloba L. leaves (1, 2, 5, 10, 15, and 20 g), concentrations of AgNO3 (1, 2, 4, and 8 mM), hydrogen potentials (pH 3, 5, 7, 9, and 11), and reaction temperatures (4, 20, 40, 60, and 80°C). UV–vis absorption spectra were measured to confirm AgNPs obtained through various conditions.

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2.4 Characterisation of AgNPs

2.5 Antifungal activity of AgNPs

Solution colour change could be observed obviously after incubation for 15 min. UV–vis spectroscopy (TU-1950), TEM (JEM-2100F), SEM (S-4800), and EDX were applied to determine AgNPs synthesised through various conditions.

On the basis of inhibition zone effect, the antifungal activity of AgNPs was examined. About 100 μl of spore suspension (106/ml) was spread uniformly on potato dextrose agar (PDA) plates. After that, 10 μl of different treatments including AgNPs, G. biloba L. filtrate, and sterile water were added to the sterile filter paper discs (5 mm×5 mm), distributed evenly on mentioned PDA plates. After standing for 5–10 min, the plates were incubated at 30°C for 2–3 days. Three replicates were performed for each treatment.

3 Results 3.1 Biosynthesis of AgNPs As shown in Fig. 1, the colour of diluted filtrate changed from (a) grey white to (b) pale yellow after incubating with 1 mM AgNO3 at 80°C for 15 min, indicating formation of AgNPs. (c) UV–vis spectrum indicated that there was a sharp absorption at 443 nm, corresponding to the surface plasmon resonance of AgNPs. 3.2 Effect of different quantities of G. biloba L. leaf The solution colour turned from light yellow to reddish brown as the leaf increased from 1 to 20 g (Figs. 2a–f). From 1 to 15 g, maximum absorption increased, while it decreased significantly at 20 g (Fig. 2g). As a result, 15 g is optimum quantity of G. biloba L. leaf for this process. The following optimisation experiments refer to this quantity, unless otherwise noted. 3.3 Effect of different concentrations of AgNO3

Fig. 1 Biosynthesis of AgNPs, based on 1 g G. biloba L. leaf

The solution colour deepened with increased AgNO3 concentration (Figs. 3a–d). Absorbance peak decreased first and increased subsequently, it reached maximum at 8 mM AgNO3 (Fig. 3e).

(a), (b) Colour change of diluted filtrate before and after addition of 1 mM AgNO3 at 80°C, (c) UV–vis spectrum of AgNPs

Fig. 2 Effect of varied quantities of G. biloba L. leaf on AgNPs formation (a)–(f) Show 1, 2, 5, 10, 15, and 20 g, (g) UV–vis spectra of synthesised AgNPs

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3.4 Effect of different reaction temperatures Increased reaction temperature is necessary for AgNPs formation, the solution colour obviously deepened as temperature increased from 4 to 80°C (Figs. 4a–e). There was no obvious absorbance peak when the temperature was below 40°C (data not shown). However, it increased gradually as temperature enhanced to 60°C or higher and maximum absorbance occurred at 80°C (Fig. 4f). 3.5 Effect of varied pH The solution pH is a critical parameter influencing formation of AgNPs. Solution presented white when its pH was 3, corresponding absorption peak was below the baseline (data not shown), while it became darker as pH increased (Fig. 5a). UV–vis spectra showed that the absorbance and pH exhibited a positive correlation to some extent, the optimal pH was 9 not 11 (data not shown). 3.6 TEM analysis of AgNPs Fig. 6 shows the typical TEM image of the synthesised AgNPs. (a) They were spherical or near spherical with favourable dispersal behaviour. To determine particle size and size distribution, 200 particles were selected from several TEM micrograms randomly. (b) Biosynthesised AgNPs were in the range of 6–32 nm and average particle size was about 14 nm. 3.7 SEM and EDX analyses of AgNPs

Fig. 3 Effect of different concentrations of AgNO3 on AgNPs formation (a)–(d) Show 1, 2, 4, and 8 mM AgNO3, (e) UV–vis spectra of synthesised AgNPs

As shown in Fig. 7a, AgNPs dispersed on the substrate as white spots when illuminated by SEM. Peaks at 3 keV indicate existence of elemental Ag, while other peaks should be due to copper grid and elements of G. biloba L. leaf extract. 3.8 Antifungal effect of AgNPs against S. turcica Fig. 8 shows strong antifungal activity of AgNPs against S. turcica. Obvious inhibition zones appeared in each plate with AgNPs based

Fig. 4 Effect of different reaction temperatures on AgNPs formation (a)–(e) Show 4, 20, 40, 60, and 80°C, (f) UV–vis spectra of synthesised AgNPs


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Fig. 5 Effect of different pH on AgNPs formation (a) pH were 3, 5, 7, 9, and 11 from left to right, (b) UV–vis spectra of synthesised AgNPs

Fig. 6 TEM analysis of AgNPs (a) TEM micrograph, (b) Size distribution of AgNPs

Fig. 7 SEM and EDX analyses of AgNPs (a) SEM micrograph, (b) EDX spectroscopy of AgNPs

on varied quantities of leaf and pH. However, there was no inhibition zone when AgNPs were replaced by sterile water or leaf filtrate. To quantify the resulting inhibition zone, diameter of each sample was measured separately. On the whole, the diameter of inhibition zone is in the range of 7.0 ± 1.41–13.0 ± 1.79, 2 g G. biloba L. leaf-mediated AgNPs with pH of 9.0 present the best inhibition effect (Table 1).

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4 Discussion Synthesis of metal NPs is a hot research field in view of their unique properties and extensive value. In this research, G. biloba L. leaf was used to synthesise AgNPs, a simple and rapid synthetic method, and it is also consistent with ‘green chemistry’. Although Song and Kim [27] reported synthesis of AgNPs by five different plants including G. biloba, the key point embodied in Diopyros kaki and Magnolia kobus, and the results mainly associated with various reaction times. About 2 months ago, Ren et al. [28] IET Nanobiotechnol., 2017, Vol. 11 Iss. 7, pp. 803-808 © The Institution of Engineering and Technology 2017

Fig. 8 Inhibition zones created by G. biloba L. leaf-mediated AgNPs against S. turcica (a)–(f) Represent quantities of leaf were 1, 2, 5, 10, 15, and 20 g. In each plate, the labels of 3, 5, 7, 9, and 11 correspond to varied pH of each AgNPs

Table 1 Diameter of inhibition zone in condition of different AgNPs Different quantities of leaves, g G. biloba L. leaf-mediated AgNPs diameter of inhibition zone, mm 3 5 7 9 11 1 2 5 10 15 20

12.3 ± 4.19 12.3 ± 4.19 11.3 ± 3.88 9.0 ± 2.77 11.7 ± 4.15 9.7 ± 3.01

reported green synthesis of AgNPs using G. biloba L. leaf extract. The authors developed high concentration of AgNO3 (20–80 mM) to synthesise AgNPs, it is worth trying that much lower concentration out of low cost concerns. In addition, in order to discover optimum synthesis system, there are other parameters that affect synthesis of AgNPs should also be measured besides concentration of AgNO3 such as quantity of leaf, concentration of AgNO3, reaction temperature, and pH. Antibacterial activity of AgNPs synthesised by G. biloba L. leaf extract was demonstrated as Ren et al. [28]. It is necessary to measure its antifungal effect on fungi that do serious harm to agriculture. Taking S. turcica as an example, G. biloba L. leafmediated AgNPs exhibit excellent inhibition effect, and mildly alkaline environment is favourable to enhance the antifungal efficacy. These results may provide a novel approach for integrative control of plant pathogenic fungi, and it also would be a valuable candidate for pathogen-resistant management.

5 Conclusion On the basis of this experiment, AgNPs were synthesised conveniently and inexpensively in short time, and the optimum synthesis system was also ascertained. Moreover, antifungal effect of AgNPs against one kind of plant pathogenic fungi was proved. Further experiments should be focused on other main phytopathogenic fungi, bacteria, and drug-resistant plant pathogens, certain factors including particle size, particle morphology, and adverse circumstance that influence inhibitory effect of AgNPs will be carried out in the near future. IET Nanobiotechnol., 2017, Vol. 11 Iss. 7, pp. 803-808 © The Institution of Engineering and Technology 2017

10.7 ± 2.57 11.0 ± 2.77 7.7 ± 1.61 10.0 ± 2.35 10.0 ± 2.35 10.0 ± 2.24

10.3 ± 1.55 11.0 ± 1.77 7.0 ± 1.41 8.0 ± 0.84 11.3 ± 2.22 10.0 ± 1.52

10.0 ± 0.84 13.0 ± 1.79 12.3 ± 1.70 12.0 ± 1.34 12.0 ± 1.52 11.3 ± 1.12

10.0 ± 0.45 10.3 ± 1.80 8.0 ± 1.52 9.3 ± 0.85 12.7 ± 0.85 11.0 ± 0.71

6 Acknowledgments This work was supported by the Natural Science Fund of Education Department of Anhui province (KJ2017A510), the Key Discipline of Plant Protection in University of Science and Technology of Anhui (AKZDXK2015C04), the Key Project of Anhui Education Department (KJ2013ZD01), and the Talent introduction project in Anhui Science and Technology University (2016).

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