Journal of Phytopathology and Pest Management 5(2): 88-107, 2018
pISSN:2356-8577 eISSN: 2356-6507
Journal homepage: http://ppmj.net/
Corresponding author:
M.A. Mohamed,
Tel: +201018274608, Fax: +20882342708,
E-mail: mohammed_sharouny@yahoo.com
88
Copyright © 2018
Antifungal activity of different size controlled
stable silver nanoparticles biosynthesized by the
endophytic fungus
Aspergillus terreus
M.A. Mohamed
1,2*
, H.M. Hussein
3
, A.A.M. Ali
4
1
Plant Pathology Research Institute, Agricultural Research Center, Giza 12655, Egypt
2
Instituto de Biología Molecular y Celular de Plantas (Consejo Superior de Investigaciones Científicas - Universidad
Politécnica de Valencia), Avenida de los Naranjos, 46022 Valencia, Spain
3
Chemistry Department, Faculty of Science, Helwan University, Ain-Helwan, Cairo 11795, Egypt
4
Plant Pathology Department, Faculty of Agriculture and Natural Resources, Aswan University, Aswan, Egypt
Abstract
Keywords: silver nanoparticles, size control, pH, biosynthesis, Aspergillus terreus, antifungal activity.
Silver nanoparticles (AgNPs) synthesized by using the aqueous extract of the
endophytic fungus Aspergillus terreus F37 (KX024595) as reducing agent is
reported here. The reaction medium employed in the synthesis process was
optimized under a narrow range of pH and temperature to attain better yield,
controlled size, and more stable of AgNPs. Further, the microbially synthesized
AgNPs were studied through UV-vis spectroscopy, transmission electron
microscopy (TEM), X-ray diffraction (XRD), and Fourier transform infrared (FT-
IR) spectroscopy analyses. The obtained results indicated the formation of high
crystalline spherical AgNPs with an average diameter of 45.2±0.5 nm at room
temperature (22 ºC). Quantitative analyses indicated that reduction of the Ag+
precursor was promoted at elevated pH due to increased activity of biomolecules in
the fungal extract. As a result, the size of the AgNPs decreased with increased pH
of the reactions. The optimum conditions for maximum production of small control
sized AgNPs (12± 0.5 nm) were pH (10) and temperature (100 ºC). The outcomes of
the antifungal activity of different controlled sized AgNPs showed their efficiently
to inhibit the mycelial growth of the pathogenic fungus Alternaria solani, the causal
agent of tomato early blight disease and reduced their viability in a pH and
temperature dependent manner. These findings revealed that the fine tuning of the
reaction synthesis parameters, will increase the chance to obtain desired well
shaped and small sized AgNPs with potent antifungal activities, may have
important applications as new bio-fungicides in controlling various plant diseases
caused by fungi.
Mohamed et al., 2018
89
Introduction
Silver nanoparticles (AgNPs) have been
widely studied during the past few
decades due to their unique optical and
electric properties and potential
applications in electronics (Evanoffand &
Chumanov, 2004), catalysis (Mohamed et
al. 2016; Mallick & Witcomb, 2006), bio-
labeling (Zhang et al., 2005; McFarland
& Van Duyne, 2003), and also and also
biotechnology (Khatami et al., 2018;
Abd-Alla et al., 2016; Kathiravan et al.,
2015; Iravani, 2011).The AgNPs are also
well known to exhibit a broad spectrum
of biocidal activity towards many
bacteria, fungi and viruses (Cordero et al.,
2017; ; Abd-Alla et al., 2016; Kumar &
Sujitha, 2014; Zachariadis et al., 2004).
Interestingly, the antimicrobial effect of
AgNPs is size dependent, where as their
size is smaller as the antimicrobial effect
is more potent (Morones et al., 2005).
Hence, extensive works have been carried
out to synthesize silver nanoparticles with
controllable shape and size desired (Lu et
al., 2006; Wiley et al., 2006; Ni et al.,
2005; Sherry et al., 2005). Reduction of
the silver salt precursor (AgNO
3
) by
chemical reductants such as citrate (Pillai
& Kamat, 2004), ascorbic acid (Sondi et
al., 2003; Velikov et al., 2003), or sodium
borohydride (Ahmadi et al., 1996) is
among the most used methods for the
synthesis of AgNPs in aqueous solution.
However, the AgNPs produced by the
citrate reduction route were usually tend
to exist in mixtures of different shaped
(e.x spherical and rod-like) due to the
poor balance of nucleation and growth
processes (Dong et al., 2009) and those
produced by sodium borohydride were
usually small spherical silver
nanoparticles (<10 nm) due to the high
reactivity of the borohydride (the
reductant) which may lead to induce the
explosive nucleation process. So, it is less
productive to tune the nucleation and
growth processes and thus the size of the
AgNPs by changing the reaction
parameters such as molar ratio of the
reductant/silver precursor, pH, or
temperature of the reactions when citrate
or sodium borohydride was employed as
the reductants in the synthesis process.
Also, involving such reductans like
borohydride in the AgNPs synthesis
cause environmental toxicity or
biological hazards which limits its
applications in human being applications.
By taking all together in parallel with
LaMer model (Ji et al., 2007), a
reasonable way to prepare silver
nanoparticles with tunable size is to
choose a reductant with suitable
reactivity and safe impact to mediate the
nucleation and growth processes of the
particles. In another hand, Qin et al.
(2010) reported a dependence between
size and pH of the reaction system, as pH
of the reaction mixture is increased, the
average size of the AgNPs was decreased
(Reddy et al., 2014). This stimulates the
scientists to search out clean, non-toxic
and environmentally acceptable
biological routes for the synthesis of
AgNPs (Escárcega-González et al., 2018;
Mittal et al., 2013; Qian et al., 2013).
Fungi are a good option compared to
other eukaryotes because of the vast
repertoire of proteins, enzymes, and other
bioactive secondary metabolites that they
produce, which possess redox capacity
and, thus, increase productivity during
the biosynthesis process (Abdel-Hafez et
al. 2016 a,b; Mohamed 2015; Phithiviraj
et al. 1998). These biomolecules serve as
reducing agents to reduce various silver
salts to its corresponding zero valent
metallic nanoparticles and also as
stabilizing agents to prevent
nanoparticles from agglomeration.
Moreover, the biosynthesis route has not
only resulted in environmental benefits,
but also enhances their physico-chemical
Mohamed et al., 2018
90
property, which leads to more effective
application (Morsy et al., 2014; Liu et al.,
2013; Tanvir et al., 2012). Although
silver nanoparticles are effective against a
number of phytopathogenic fungi
including
Bipolaris sorokiniana
and
Magnapothe grisea
(Shrivastava et al.,
2007; Panacek, et al., 2006; Morones et
al., 2005), however many
phytopathogenic fungi are not explored
although they are causing destructive
diseases on important crop plants and
thereby reducing the yield of agricultural
products.
Alternaria solani
(Ellis &
Martin) Jones & Grout is a soil
inhabiting, air-borne fungal pathogen
responsible of tomato early blight
disease, one of the most important and
frequent fungal disease infecting tomato
crops worldwide, causing reduction in
tomato crop quantity and quality (Song
et al., 2011). Our aim in the present work
was to biosynthesize AgNPs using the
endophytic fungus
Aspergillus terreus
F37 (KX024595), to characterize them
under a range of pH and temperature
values and to test their antifungal activity
against three different pathogenic isolates
of
Alternaria solani
the causal agent of
tomato early blight disease.
Materials and methods
Sample collection and isolation of
endophytic fungi:
Four hundreds of
fresh healthy tomato (
Solanum
lycopersicum
L.) leaf plants were
collected from Egypt in 2015 to isolate
endophytic fungi.
The collected samples
were transferred directly to the
mycological laboratory, Botany and
Microbiology Department, Assiut
University, Egypt. At laboratory, leaves
were gently washed with running tap
water and aseptically cut into small
segments (5 × 5 mm). All segments were
rinsed with distilled water and surface
sterilized following the sequence: 70%
ethyl alcohol for one minute, and then
transferred to a solution of 2.5% sodium
hypochlorite for 3.5 min, followed by a
treatment with 70% ethanol for 30 s. the
prepared segments were then put on Petri
dishes containing potato dextrose agar
(PDA) medium containing 250 mg L
-1
streptomycin and incubated at 26 ± 2 °C
for 8 days. The hyphal tips of the fungal
endophytes growing out from the plant
tissues were carefully transferred onto
new PDA plates under sterilized
conditions and incubated at 26 ± 2 °C for
8 days. After incubation, fungal
endophytes identification was performed
according to morphological
characteristics. Percent colonization
frequency (% CF) of endophytic fungi
was calculated according to Petrini and
Fisher (1988): colonization frequency
(%) = (total number of segments
colonized/ total number of segments) ×
100.
DNA extraction:
The frozen mycelia of
the fungus (100 mg) were ground with
liquid nitrogen in a mortar and pestle and
mixed with 1 ml of 4 M guanidinium
thiocyanate, 0.1 M sodium acetate pH
5.5, 10 mM ethylenediaminetetraacetic
acid (EDTA), 0.1 M 2-mercaptoethanol.
Extracts were clarified by centrifugation
and supernatants were loaded into silica
gel spin columns (Wizard Plus SV
Minipreps DNA Purification, Promega,
USA). Columns were washed with 70%
ethanol, 10 mM sodium acetate pH 5.5,
and DNA eluted with 50 µl of 20 mM
Tris-HCl, pH 8.5.
Ribosomal DNA amplification and
sequencing:
A ribosomal internal