Biosynthesis of AgNPs by B. maydis and its antifungal effect against Exserohilum turcicum

Abstract

In the present study, Bipolaris maydis was used to synthesise silver nanoparticles (AgNPs). Several parameters that influence the synthesis of AgNPs such as fungus age, the concentration of Ag nitrate (AgNO₃), and incubation time were explored to find the optimum synthesis condition. Furthermore, the antifungal activity of AgNPs against Exserohilum turcicum was determined by measuring inhibition zone diameter, colony formation, and conidia germination. The optimal biosynthesis system included fungus age of 7 days, 8 mM AgNO₃, and an incubation time of 120 h. Under these conditions, synthesised NPs were near round, and the average particle size was about 21 nm. At the experiment, the diameter of the inhibition zone reached a maximum of 8 mM AgNO₃ and 72 h. In addition, the inhibition rate of colony and conidia reached 83.39 and 100%, respectively, with 200 μg/ml AgNPs. The results offer a novel pathway for phytopathogen control and make it likely to develop new eco‐friendly antimicrobial.

1 Introduction

The importance of nanotechnology has been shown by extensive applications in the present society. Conventional materials are facilitated into nanoscale, 1–100 nm at least in one dimension [1]. On the nanoscale level, materials have different chemical, physical, magnetic, electrical, and optical properties compared with their bulk counterparts due to their high specific surface area [2].

Silver nanoparticles (AgNPs) have drawn the attention of an increasing number of researchers in the fields of medicine, electronic, optics, and chemistry [3, 4]. Synthesis of NPs can be achieved via different methods such as ultraviolet (UV) irradiation [5], microwave‐assisted [6, 7], electrochemical [8], sonochemical [9], microfluidic route [10], microorganism‐mediated [11, 12, 13, 14, 15, 16], and plant extract‐mediated methods [17, 18, 19, 20, 21, 22]. Physical methods consume energy, while chemical methods contain toxic chemicals; therefore, it is necessary to advocate green synthesis of NPs. Production of NPs based on microorganism and plant extract confirm to the concept of green chemistry. During the past years, various living organisms were applied to synthesise AgNPs; for example, Bacillus koriensis [11], Bacillus sp. [15], Fusarium oxysporum [23], Trichoderma viride [13], Aspergillus fumigatus [16], Septoria apii [14], Pulicaria gnaphalodes [24], Caulerpa racemosa [25], Brassica rapa L. [26], Ginkgo biloba [27], and Osmanthus fragrans [28]. In recent years, animal species such as arthropods and metabolites [29], bio‐resources as agro‐wastes, enzymes, and pigments were also used to synthesise AgNPs [30].

Ag was considered as a bacteriostatic agent in the 19th century. Afterwards, antimicrobial, antioxidant, anticoagulant, and thrombolytic activity of Ag and relevant products were extensively measured [29, 30]. Among these forms, nanoscale particles proved to be the most effective. It has been reported that AgNPs synthesised by date palm pit aqueous extract exhibited prominent antifungal effect at 83% at 25 μg/ml against Descurainia sophia, and minimum bactericide concentration against Klebsiella pneumonia and Acinetobacter baumannii were recorded at 1.56 and 3.12 µg/ml [31]. Khatami et al. reported that AgNPs synthesised by D. sophia showed a maximum inhibitory effect against Rhizoctonia solani at 86% at 25 μg/ml, and the reported minimum bactericidal concentration of AgNPs against Agrobacterium tumefaciens and Agrobacterium rhizogenes were 4 and 8 μg/ml, respectively [32]. Xia et al. [33] evaluated the effect of AgNPs against Trichosporon asahii and the minimum inhibitory concentration was 0.5 μg/ml. Nejad et al. [34] applied green‐synthesised AgNPs to interact with R. solani and the highest inhibition levels against sclerotia formation and mycelia growth were 92 and 85%, respectively, at a concentration of 50 ppm. Swamy and Nargund [35] measured the antifungal effect of AgNPs against Curvularia lunata, and it was found that more than 95% of spores were inhibited at the tested concentration.

In this research, Bipolaris maydis was used to biosynthesise AgNPs, and the optimisation of AgNPs formation was achieved through regulating the incubation time of the isolate, the concentration of Ag nitrate (AgNO₃), and the fungus age. The synthesised AgNPs were characterised using UV–visible (UV–vis) spectrophotometry, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy dispersive X‐ray (EDX). Furthermore, the antifungal effect of AgNPs was measured through inhibition zone, colony formation, and conidia germination against Exserohilum turcicum.

2 Materials and methods

2.1 Microorganism isolation

The fungus used here for AgNPs production was B. maydis, and the fungus used for antifungal effect was E. turcicum, both of which were conserved in the Plant Protection laboratory of the Anhui Science and Technology University.

2.2 Preparation of hyphae filtrate

The hyphae filtrate was prepared as follows: B. maydis was grown on Potato Dextrose Agar (PDA) plates at 30°C for 3–15 days. Subsequently, it was inoculated into Potato Dextrose (PD) liquid medium, followed by shaking at the speed of 150 r/min for more than 24 h. Then, the liquid cultures were filtered through sterile filter papers, and the filtrate was conserved at 4°C.

2.3 Optimal biosynthesis of AgNPs

AgNPs was synthesised by appending the obtained filtrate to deionised water at a specific rate of 1:9 (v:v); different concentrations of AgNO₃ was followed. This confirmed the formation of AgNPs as the solution colour changed. Several parameters were amended to optimise synthesis process including different spawn ages (3, 7, and 15 days), concentrations of AgNO₃ (1, 2, 4, and 8 mM), and co‐cultivation time (12, 24, 48, 72, 96, and 120 h). AgNPs synthesised through such conditions were recorded via digital images and UV–vis absorption spectra.

2.4 Characterisation of AgNPs

Co‐cultivation of diluted filtrate and AgNO₃ was selected every 24 h to measure the absorption spectra via UV–vis spectroscopy (TU‐1950). The solution obtained via the optimal synthesis process was applied to scan the morphology using TEM (JEM‐2100F) after totally drying on the copper grid. For SEM (S‐4800) analysis, such synthesised AgNPs were deposited on a sample plate, followed by platinum coating. EDX was utilised to analyse the components and their contents in the solution.

2.5 Antifungal effect of AgNPs

2.5.1 Inhibition zone assay

Antifungal activity of AgNPs against E. turcicum was implemented by measuring the diameter of the inhibition zone. About 0.1 ml of spore suspension (10⁶ /ml) was uniformly smeared on solidified PDA plates. Then, 7 μl of various samples such as AgNPs, sterile water, and filtrate was dripped onto the fixed sterile filter paper discs (5 mm×5 mm). After 5 min of standing, the plates were incubated at 30°C for 3 days. Three replicates were conducted per treatment.

2.5.2 Inhibition of colony growth

Oven‐dried AgNPs were dissolved in sterile water as the stock solution (10 mg/ml). A volume of 5 ml of diluted stock solution was added to 45 ml of PDA medium at an approximate temperature of 55–60°C, and final concentrations of AgNPs of 6.25, 12.5, 25, 50, 100, and 200 μg/ml were obtained via dilution with sterile water. The control set contained 5 ml of sterile water without AgNPs. A fungus block (φ  = 5 mm) was inoculated in the centre of each Petri dish containing different concentrations of AgNPs, followed by incubation at 28°C for 3–5 days. Each control and experimental treatment was performed in three replicates.

2.5.3 Influence on conidia germination

The densities of E. turcicum conidia were adjusted to 10⁶ /ml via counting chamber. Next, various concentrations of AgNPs (6.25, 12.5, 25, 50, 100, and 200 μg/ml) and conidia suspensions were added into sterile centrifuge tubes at a ratio of 1:9 (v:v), the conidia suspension served as control. Then, incubation at 25°C for 1–2 days was performed. Images of conidia in the control and experimental treatments were obtained under a microscope (100×).

3 Results

3.1 Formation of AgNPs

As shown in Fig. 1, the colour of diluted filtrate changed from light green (a) to reddish brown (b) after incubating with 1 mM AgNO₃ at 30°C for 96 h, denoting synthesis of AgNPs. The UV–vis spectrum showed a strong and broad surface plasmon resonance peak at 430 nm (c), which is characteristic for AgNPs.

Fig. 1.

Synthesis of AgNPs by B. maydis

(a) Digital image of diluted filtrate without AgNO₃, (b) Digital image of filtrate with 1 mM AgNO₃, (c) UV–vis absorption spectrum of AgNPs

3.2 Optimal biosynthesis of AgNPs

3.2.1 Effect of fungus age

Taking a certain concentration (2 mM) of AgNO₃ as an example, the co‐culture of AgNO₃ and B. maydis (3–15 days) appeared reddish brown compared with the filtrate, and there were no obvious differences in Figs. 2 a –c. However, UV–vis spectra of AgNPs based on different fungus age varied. Overall, maximum absorption decreased along with increasing fungus age. Only from the point of maximum absorption peak, the maximum was reached for the isolate of 3 days; however, the corresponding absorption wavelength was 350 nm, which conflicted with the surface plasmon resonance of AgNPs. Therefore, the isolate of 7 days was optimised to synthesise AgNPs.

Fig. 2.

Effect of varied fungus age on AgNPs formation

(a)–(c) Digital photographs of AgNPs with fungus ages of 3, 7, and 15 days, (d) , UV–vis spectra of synthesised AgNPs based on different fungus ages

3.2.2 Influence of various concentrations of AgNO₃

The solution colour darkened via gradually increasing the concentration of AgNO₃ (Fig. 3 a). Correspondingly, the absorption peak increased continuously, and a maximum was achieved at 8 mM AgNO₃ (Fig. 3 b).

Fig. 3.

Influence of various concentrations of AgNO₃ on AgNPs synthesis

(a) Digital photographs of AgNPs with 1, 2, 4, and 8 mM AgNO₃, (b) UV–vis spectra of synthesised AgNPs based on different concentrations of AgNO₃

3.2.3 Effect of different incubation times

Under the condition of 8 mM AgNO₃, the solution colour of AgNPs deepened compared with those that contained low concentrations of AgNO₃, and it became dark brown when the incubation time reached 120 h (Fig. 4 b). As shown in Fig. 4 c, the maximum absorption of AgNPs decreased as the incubation time prolonged from 24 to 120 h, reaching a minimum at 96 h.

Fig. 4.

Effect of different incubation times on AgNPs formation

(a), (b) Synthesised AgNPs based at 24 and 120 h, (c) UV–vis spectra of synthesised AgNPs based on different incubation times

3.2.4 TEM detection of AgNPs

Fig. 5 showed a representative TEM image of the synthesised AgNPs. The particles were round or near round with favourable dispersibility (Fig. 5 a). To define particle size and size distribution, 200 particles were randomly chosen from several TEM images. Biosynthesised AgNPs were confined to be 8–41 nm with an average particle size of about 21 nm (Fig. 5 b).

Fig. 5.

TEM detection of AgNPs

(a) TEM micrograph, (b) Size distribution of AgNPs

3.2.5 SEM and EDX analyses of AgNPs

Fig. 6 a showed quantities of particles deposited on the substrate. Peaks near 3 kev indicate Ag, and the remaining peaks should be attributed to copper grid and elements of fungus filtrate.

Fig. 6.

SEM and EDX analyses of AgNPs

(a) SEM micrograph, (b) EDX spectroscopy of AgNPs

3.2.6 Inhibition zone assay

Fig. 7 shows strong antifungal effect of AgNPs against E. turcicum. Apparent inhibition zones emerged in the plates with AgNPs based on different incubation times and concentrations of AgNO₃. However, no inhibition zone was found when the sample contained sterile water or fungus filtrate.

Fig. 7.

Inhibition zones formed by AgNPs against E. turcicum

(a)–(e) Incubation times of 48, 72, 96, and 120 h, respectively. In each plate, the label of 0 and 1 correspond to sterile water and fungus filtrate; 2, 3, 4, and 5 represent different concentrations of AgNO₃ as 1, 2, 4, and 8 mM

To see the result more clearly, the diameter of inhibition zone was measured via crossing method. Taking a certain fungus age (7 days) as an example, this ranged between 5.0 ± 0.84 and 10.7 ± 1.26 mm. The maximum corresponded to AgNPs based on incubation time of 72 h and 8 mM AgNO₃ (Table 1).

Table 1.

Diameter of inhibition zone under condition of various AgNPs

Different incubation times, h	Biosynthesised AgNPs diameter of inhibition zone, mm
	1	2	4	8
24	6.0 ± 0.84	7.2 ± 2.32	8.5 ± 2.48	10.2 ± 0.99
48	6.2 ± 0.51	6.8 ± 2.23	8.5 ± 2.04	9.5 ± 0.76
72	7.2 ± 2.85	7.7 ± 2.57	8.7 ± 2.13	10.7 ± 1.26
96	6.0 ± 1.52	7.2 ± 2.37	7.7 ± 1.69	9.7 ± 0.85
120	6.2 ± 1.94	6.7 ± 2.13	8.3 ± 1.98	9.7 ± 0.85

3.2.7 Inhibition of colony growth

As shown in Fig. 8, AgNPs exhibited a prominent inhibition effect on colony growth. The diameter of E. turcicum without AgNPs measured via the cross‐method was 6.10 cm (Fig. 8 a), and it decreased gradually with increasing concentration of AgNPs (Figs. 8 b –g). The diameter reached a minimum value of 1.43 cm at 200 μg/ml of AgNPs (Fig. 8 g). The inhibition rate caused by the varied AgNPs concentrations (6.25–200 μg/ml) in the control and experimental treatments of E. turcicum was in the range of 17.86–83.39%.

Fig. 8.

Inhibition of colony growth of E. turcicum by AgNPs

(a) Colony of the control isolate, (b)–(d) E. turcicum with AgNPs concentrations of 12.5, 25, and 200 μg/ml, respectively

3.2.8 Influence of conidia germination

As seen from the data in Fig. 9, AgNPs dramatically affected the germination of conidia of E. turcicum. In the control, the germination rate was 93.94%; however, it decreased notably with the increase in the concentration of AgNPs, and conidia were totally inhibited at 200 μg/ml.

Fig. 9.

Influence of AgNPs on conidia germination of E. turcicum

4 Discussion

The production of metal NPs, especially of AgNPs, is a popular field, considering the particular properties and broad application values. In this experiment, B. maydis was used to synthesise AgNPs without using extra toxic chemical reagent, and the result is in accordance with ‘green chemistry’. Although several review papers exist about AgNPs synthesised by fungi [11, 29, 30], B. maydis has not been adopted to date, and an optimal synthesis system was determined through adjusting fungus age, the concentration of AgNO₃, and incubation time for the first time. In addition, compared with AgNPs synthesised by other fungi reported in previous studies, several differences were found such as solution colour [14, 36], particle shape [36], particle size and size distribution [14, 16, 36], maximum absorption wavelength [36, 37] etc. The reasons for such differences might be fungus species, fungus age, AgNO₃ concentration, and incubation time.

Antifungal activity showed that AgNPs synthesised by B. maydis exhibit excellent inhibition effect against E. turcicum. On the basis of existing results, this showed an apparent discrepancy compared with other relevant reports. It has been reported that the antifungal effect of the same AgNPs against different pathogens differentiated [16, 25, 37]. AgNPs synthesised through different methods, microorganisms or plants showed different inhibition activity against the same pathogen [31, 32, 34]. In addition, the antimicrobial activity of AgNPs was influenced by other parameters such as the pH of the solution [28], the stabiliser [38], and the modified substances [19, 30]. These results may offer a novel approach for the comprehensive control of phytopathogens, and it also avoids pathogen resistance to the utmost extent.

5 Conclusion

On the basis of these results, one type of important phytopathogen called B. maydis that caused severe damage to maize could synthesise AgNPs, and several parameters which influence its synthesis were conducted to ascertain the optimal condition. In addition, as‐synthesised AgNPs showed prominent antifungal activity against E. turcicum including inhibition zone, colony formation, and conidia germination. In the near future, experiments should focus on parameters that affect antifungal activity such as aggregation, charge, UV ray, and high temperature.