Abstract
(NPs) can be produced by various methods such as physical and chemical processes. However, environmentally friendly ways are increasingly requested. In this research, (Ag-NPs) were produced by Fusarium oxysporum, and its antifungal effect on Aspergillus and Fusarium was investigated. Nanoparticles were produced by silver nitrate salt and Fusarium oxysporum native to Isfahan city. In order to optimize the synthesis conditions, optimization of some factors such as volume, concentration, time, temperature, and pH of the extract was performed. The structural and physical properties of NPs were determined by spectrophotometer, XRD, FTIR FESEM, SEM, and TEM microscopy. For the study of the inhibitory effect of NPs on Fusarium and Aspergillus growth, the fungi were cultured in media containing various concentrations of NPs from 50 to 1500 ppm. Then, the colony diameter was measured for over 10 days and the growth inhibition percentage was estimated. For statistical analysis, the 600 Mann–Whitney tests have been applied.The NPs were produced after mixing the powdered fungal mass and silver nitrate salt in optimum conditions which were 2 mM of salt, triple fungal mass volume proportion relative to the salt, pH of 9, and temperature of 28 °C. The existence of a peak at 420 nm in FTIR was due to nanoparticle production. Based on the XRD, the synthesized NPs had suitable properties similar to the standard NPs reported in the studies. Images from TEM, SEM, and FESEM microscopes displayed uniform NPs in variable sizes between 25 and 100 nm. According to the results, the maximum growth inhibition percentage of Ag-NPs on Fusarium was approximately 60% at 1500 ppm, and 88% on Aspergillus at 800 ppm. Biosynthesized Ag-NPs with Fusarium oxysporum have desirable structural traits and can inhibit the growth of Fusarium and Aspergillus at significant levels.
Keyword: Silver nanoparticle synthesis, Fusarium oxysporum.antifungal activity
Introduction
Nanoparticles (NPs) are solid colloidal particles with a size range of about 1–100 nm. Particle properties depend on the size of the particles. At the nanometer sizes, the particles acquire unique electronic, magnetic, optical, and catalytic properties and increase the surface-to-volume ratio [1]. It is expected that nanotechnology will have wide impacts on various aspects of human life in the coming decades, including pharmaceutical and medical sciences. One of the important areas of research in nanotechnology is the synthesis of different NPs [2]. Presently, the growing need for reliable methods for the synthesis of NPs that do not harm the environment, has led researchers to work on biological systems [3]. Many organisms, either unicellular or multicellular, are known to produce inorganic NPs that act either intracellularly or extracellularly. The most important NPs are metal NPs that have significant applications in various sciences [4]. It has been shown that microorganisms, in addition to being resistant to metals, can produce metal particles on a nanoscale [5]. Silver nanoparticles (Ag-NPs) have a high surface area, small size, and desirable dispersibility. These particles, which are increasingly used in the fight against infections, are now widely used in the medical industry [6]. Ag-NPs have been used in medical sciences for the treatment of burns, dental productions, metal coatings, water treatments, sunscreen solutions, and disinfectants [7]. The most important features of Ag-NPs include non-toxicity, high stability, hydrophilicity, thermal resistance, and increased resistance to microorganisms [8].
Fusarium oxysporum is one of the pathogenic plant fungi that grow in soil all around the world. In addition, some of its strains are used as biocontrol agents in agriculture. Numerous articles have so far been published to prove that this microorganism is capable of producing metallic NPs in different forms [2].
Nowadays there is an increasing need for high-efficiency, low-cost, non-toxic, and environmentally non harmful antifungul methods. Things like plants, algae, molds, yeasts, bacteria, and viruses are used in the bio-production of NPs [4–6]. The use of multi enzymes containing fungi has been the focus of today's synthesis of NPs. The secretion of the enzyme into the extracellular space provides the appropriate conditions for these enzymes to be used in the synthesis of NPs [9]. It provides access to the appropriate enzymes, away from other cellular proteins and, without the need for filtration. This access is an important and appropriate reason for the use of fungi in nanoparticle synthesis. This process which suits the identification of enzymes secreted by fungi can be expanded to the synthesis of NPs of different chemical constituents, shapes, and sizes [9, 10]. Understanding the surface chemistry of biogenic NPs such as surfactants, peptides, and proteins, is also important. This enables the genetic engineering of microbes to overproduce the specific reducing molecules and the limiting factors that control the size and shape of NPs [8]. The logical use of confined space within cells, such as the periplasmic and cytoplasmic vesicular compartments, has led to control the size and shape of NPs, but their isolation has not been doable yet [6–8]. In the present age, fungi are a serious threat to human, animal and agricultures. Considering the importance of bio-production of NPs, the present study will investigate the synthesis of Ag-NPs by Fusarium oxysporum and its performance against Aspergillus fumigatus and Fusarium oxysporum.
Materials and Method
Isolation and Preparation of Fungi
For the synthesis of nanoparticles, Fusarium oxysporum fungi isolated from Isfahan soil. The isolated fungi were cultured in Sabouraud Dextrose Agar (SDA) medium and stored at 28 °C for 5 days.
Metal Nanoparticles
Silver nitrate salt (AgNO3) is a precursor to many compounds containing silver. To create sufficient cell biomass, the fungus was transferred from SDA medium to a liquid medium containing 3 g/l yeast extract, 3 g/l malt extract, and 10 g/l glucose, and stored at 24 ± 1 °C for 20 days. The cultured and enriched fungi were filtered through filter paper three times and then filtered with a 0.4 filter. Silver nitrate salt with concentrations of 1, 1.5, and 2 mM was prepared and 10 g of dry biomass was added to each. The obtained compound was incubated for 24 h in a shaker incubator at 28 °C and after 24 h the solution was filtered and examined with a UV spectrophotometer to ensure the presence of the NPs. To produce the powder of the NPs, a mixture of fungal mass and silver nitrate salt (nanoparticulated and discolored) was centrifuged at 14,000 rpm for 5 min [11]. The supernatant was then discarded and the final precipitate was poured into the watch glass to dry. In order to obtain the best volume of fungal biomass and silver nitrate solution, appropriate concentrations, time, and pH were investigated.
Verification of Biologically Produced Nanoparticles
Characterization of Ag-NPs is necessary for controlling and understanding of its applications and synthesis. To this end, a variety of different techniques can be used, including transmission and scanning electron microscopy (TEM and SEM), dynamic light scattering (DLS), powder X-ray diffractometry (CARD), Fourier-transform infrared spectroscopy (FTIR), and UV–vis spectroscopy [8]. These are for the determination of fractal dimensions, particle size, shape, pore size, surface area, and crystallinity. To confirm the production of Ag-NPs, it should be observed at 420 nm wavelength. Therefore, Spectrophotometer diagrams were drawn from different fungal masses in the nanoparticle production. Morphology and surface topography of Ag-NPs were investigated by scanning electron microscopy (SEM), and the size of Ag-NPs was investigated by field emission scanning electron microscopy (FESEM). X-ray diffraction (XRD) method was used to determine the nature of the produced Ag-NPs and also to estimate the relative size of the resulting NPs. FTIR experiments were performed to achieve the factors involved in nanoparticle synthesis by adding silver nitrate salt to the fungal mass. Information about size, distribution uniformity, and morphology of NPs was analyzed using TEM.
Antifungal Activity
Fusarium oxysporum and Aspergillus fumigatus were purchased and cultured from Iranian PTCC with No. PTCC = 5115 (CB 5620.87) and No. PTCC = 5158, respectively. The powdered NPs were weighed in different amounts and poured into the solvent, distilled water, sonicated for 30 min and poured into wells containing culture medium. After mixing the contents of each Erlenmeyer, 20 ml of that the solutions were scattered on each plate, the Fusariumoxysporum was inoculated on the plates, and the appropriate volume was chosen. Then, after specifying that equal volumes of NP and sabouraud dextrose agar solution is the appropriate mixture, 20 ml of the culture medium was poured into each plate containing 10 ml of the desired nanoparticle solution and 10 ml of the sabouraud dextrose agar. Each plate had a different ppm of the nanoparticle. The percentage of growth inhibition in each case was calculated using the following formula and this experiment was repeated three times.
Control colony diameter-treated colony diameter/Control colony diameter × 100
All of these steps were performed for Aspergillus fumigatus and Amphotericin B treatments and growth inhibition percentage was calculated for them. Amphotericin B was compared with the synthesized NPs at concentrations of 200, 400, and 600 ppm.
Results
Bioproduction of nanoparticles is known as a green way to produce them. The use of fungi is one of the most common bioproduction methods. After adding the silver nitrate solution to the fungal extract, the enzymes and proteins in the Ag+ extract react with the ions to neutralize them. This reaction causes discoloration in the reaction mixture and refers to the extracellular synthesis of the NPs.
Optimization of Factors Involved in Biological Silver Nanoparticle synthesis
Optimization of factors involved in silver nanoparticle synthesis, including fungal mass volume, concentration, pH, and time, was done biologically with Fusarium oxysporum. Nanoparticle synthesis was performed in sample 1 (equal volume) and sample 2 (triple of salt volume). Equal volumes of fungal mass and silver nitrate salt were used for subsequent steps. Due to the importance of the time factor, 2 mM of silver nitrate salt was selected for synthesis. A peak at 420 nm detected from each sample using a wavelength spectrophotometer demonstrates the synthesis of silver NPs.This peak was observed at pH = 9, which is close to the pH of the fungus biomass. Therefore, the desired pH was set equal to 9 and used for the optimization of the time factor after the preparation of 2 mM of silver nitrate salt and adding the fungal mass at different times of spectroscopy. At zero hour, no peak was observed. After 8 h, a peak was observed at 390 nm. After 12 h the peak was at 413 nm and finally, after 24 h the peak reached 420 nm, which was desirable (Fig. 1).
Fig. 1.
Spectroscopy of synthesized silver nanoparticles
Results of Spectrophotometry
The results of post-production nanoparticle spectrophotometer analysis showed a distinct peak at 420 nm, which was an early and strong verification of the accuracy of silver nanoparticle production (Fig. 2).
Fig. 2.
The existence of peak at 420 nm wavelengths shows the production of nanoparticles
Results of SEM and FESEM
The morphology and surface topography of Ag-NPs were investigated by electron microscopy. The particles were round and without aggregation, and the size of Ag-NPs was investigated by FESEM. Particle sizes were reported from 25 to 100 nm (Fig. 3).
Fig. 3.
SEM and FESEM electron microscope
Results of XRD
The results of this test showed different peaks, both related to the NPs and their associated contaminants. According to Fig. 2, XRD patterns of Miller indices at angles 111, 200, 220, and 311 correspond to angles of 38.143°, 46.255°, 64.51°, and 77.011° respectively, which confirm the existence of Ag-NPs in the biosynthetic method. The presence of silver atoms was verified by energy-dispersive X-ray spectroscopy (Fig. 4).
Fig. 4.
XRD pattern of silver nanoparticles with the biosynthesis method by Fusarium oxysporum
Results of FTIR
FTIR experiments were performed to achieve the factors involved in nanoparticle synthesis by adding silver nitrate salt to the fungal mass. The results are shown in Table 1. In this study, the IRPal.02 software was used to investigate FTIR bonds. The results of these investigations on the FTIR spectrum of the synthesized NPs are presented in Fig. 5. The results showed a strong and broad peak at 3500 cm−1 corresponding to an N–H bond of Amide–Amine. The narrow peak at 3391.51 cm−1 corresponds to the C–H Alkane bond. The strong narrow peak at 2843.83 cm−1 shown in the diagram corresponds to the C–H bond pattern. The presence of narrow strong peaks at 2423.07 cm−1 is related to the C=C Alkenes bond. At 1640.15 cm−1 the observed peak corresponds to the C–H bond. Also, the existence of N–C at 1384.65 is related to Amine-Amide bonds and the strong bond observed at 720.54 cm−1 corresponds to the C–H bond.
Table 1.
FTIR spectra of synthesized nanoparticles
| 3500 | Amine–Amide | N–H |
| 3391/51 | Alcanes | C–H |
| 2843/83 | Alcanes | C–H |
| 2423/07 | Alcenes | C=C |
| 1640/15 | Alcenes | C–H |
| 1384/6565 | Amine–Amide | N–C |
| 720/54 | Aromatic ring | C–H |
Fig. 5.
FTIR pattern of silver nanoparticles synthesized by Fusarium oxysporum
Results of TEM
The results of this microscope provide information on the size, distribution uniformity, and morphology of the NPs. The only way to determine the size of a particle is to observe and measure its size individually. After performing this procedure on the NPs, the images showed that the particles were of appropriate size to be used for biological purposes, and did not have abnormal aggregation and adhesion. Most of the particles were spherical and distributed cumulatively or dispersedly (Fig. 6).
Fig. 6.
TEM Images of the resulting AgNPs on A 120 nm scale, B 150 nm scale, and C 200 nm scale
Results of Inhibitory Effects of Synthesized Silver Nanoparticles on Fusarium Oxysporum
Culture media with different concentrations of synthesized NPs were prepared and the Fusarium oxysporum fungi were cultured at their center. Then, the colony diameters were measured from day 1 to day 10. At concentrations of 50 to 1500 ppm, the average growth inhibition percentage from day 2 to day 10 was decreasing (Tables 2, and 3). Also, with a daily increase of nano concentration, the average percentage of growth inhibition was rising. In order to compare the percentage of growth inhibition between day 2 and day 10 at different nano concentrations, the Kruskal–Wallis test, and Mann–Whitney test were used. Table 3 shows the average percentage of growth inhibition on different days. Meaningful differences between days were indicated with different letters. Based on the results of these tests, the percentage of growth inhibition on the second, third, and fourth days did not have a meaningful difference. The results showed that the greatest effect of NPs on Fusarium oxysporumwas was between day 5 to day 10 (P < 0.05; compared to.
Table 2.
Growth of F. oxysporum (colony diameter, mm) on SDA medium with different concentrations of the synthesized AgNPs for 10 days
| Concentrations (ppm) | Days | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
| 0 (control) | 4.10 ± 0.17 | 19.65 ± 0.55 | 42.43 ± 1.55 | 53.33 ± 1.53 | 68.53 ± 1.56 | 79.67 ± 0.58 | 88.00 ± 1.75 | 99.00 ± 1.00 | 99.00 ± 1.00 |
| 50 | 2.07 ± 0.12 | 11.33 ± 0.58 | 20.67 ± 1.15 | 34.67 ± 0.58 | 49.67 ± 0.58 | 62.33 ± 1.53 | 71.00 ± 1.00 | 78.67 ± 1.53 | 81.00 ± 1.00 |
| 100 | 1.07 ± 0.12 | 10.67 ± 0.58 | 19.67 ± 1.53 | 34.67 ± 1.53 | 45.33 ± 0.58 | 58.67 ± 1.15 | 68.67 ± 1.15 | 75.67 ± 1.15 | 83.67 ± 1.15 |
| 150 | 1.07 ± 0.12 | 7.33 ± 0.58 | 19.67 ± 0.58 | 29.67 ± 0.58 | 38.00 ± 2.00 | 48.67 ± 1.53 | 59.33 ± 1.15 | 68.67 ± 1.53 | 76.00 ± 1.73 |
| 200 | 1.07 ± 0.12 | 6.33 ± 0.58 | 14.67 ± 0.58 | 26.00 ± 1.00 | 31.67 ± 1.53 | 40.33 ± 0.58 | 49.00 ± 1.00 | 54.33 ± 1.15 | 60.67 ± 1.15 |
| 400 | 1.07 ± 0.12 | 3.33 ± 0.58 | 10.33 ± 0.58 | 19.67 ± 0.58 | 24.67 ± 0.58 | 31.33 ± 1.53 | 39.00 ± 1.00 | 46.33 ± 1.53 | 54.33 ± 1.15 |
| 600 | 1.33 ± 0.58 | 7.67 ± 1.15 | 15.33 ± 0.58 | 24.00 ± 1.00 | 31.33 ± 1.15 | 37.68 ± 1.58 | 41.33 ± 1.58 | 51.00 ± 1.73 | |
| 800 | 1.00 ± 0.00 | 4.67 ± 0.58 | 11.00 ± 1.00 | 20.67 ± 1.15 | 25.67 ± 1.15 | 30.33 ± 0.58 | 39.67 ± 0.58 | 47.00 ± 1.73 | |
| 1000 | 1.33 ± 0.58 | 5.33 ± 0.58 | 11.33 ± 1.15 | 19.67 ± 0.58 | 25.33 ± 0.58 | 30.67 ± 1.15 | 36.00 ± 1.00 | 45.33 ± 0.58 | |
| 1500 | 0.50 ± 0.58 | 1.33 ± 0.58 | 5.33 ± 0.58 | 11.00 ± 1.00 | 20.00 ± 2.00 | 24.33 ± 1.15 | 33.67 ± 1.15 | 41.00 ± 1.73 | |
Data are presented as mean ± SD of three replications
Table 3.
Mean growth inhibition of F. oxysporum (colony diameter, mm) on SDA medium with different concentrations of the produced AgNPs for 10 days, as a percent of the negative control
| Concentrations (ppm) | Days | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
| 50 | 49.33 ± 1.15 | 42.33 ± 2.52 | 50.67 ± 3.51 | 34.33 ± 1.53 | 27.00 ± 2.65 | 21.33 ± 1.15 | 20.00 ± 1.00 | 21.33 ± 1.53 | 19.00 ± 1.00 |
| 100 | 75.00 ± 0.00 | 45.67 ± 1.15 | 54.33 ± 4.04 | 34.33 ± 2.52 | 33.00 ± 1.73 | 26.00 ± 1.00 | 22.33 ± 2.89 | 24.33 ± 1.15 | 16.33 ± 1.15 |
| 150 | 74.00 ± 1.73 | 63.33 ± 2.89 | 53.00 ± 1.73 | 44.00 ± 1.00 | 44.00 ± 4.00 | 38.33 ± 1.53 | 33.67 ± 2.33 | 31.33 ± 1.53 | 24.00 ± 1.73 |
| 200 | 74.33 ± 1.15 | 67.67 ± 2.52 | 64.67 ± 1.53 | 50.33 ± 0.58 | 53.33 ± 3.28 | 49.33 ± 1.15 | 44.33 ± 2.08 | 45.67 ± 1.15 | 39.33 ± 1.15 |
| 400 | 74.33 ± 1.15 | 83.00 ± 2.65 | 75.33 ± 0.58 | 62.67 ± 0.58 | 63.13 ± 1.15 | 60.33 ± 2.07 | 55.67 ± 1.15 | 53.67 ± 1.53 | 45.67 ± 1.15 |
| 600 | 93.00 ± 2.65 | 81.67 ± 3.21 | 70.67 ± 1.53 | 64.33 ± 0.58 | 61.33 ± 1.15 | 56.00 ± 2.00 | 58.67 ± 0.58 | 49.00 ± 1.73 | |
| 800 | 94.67 ± 0.58 | 88.33 ± 1.53 | 78.67 ± 2.08 | 69.33 ± 2.08 | 67.00 ± 1.73 | 65.33 ± 0.58 | 60.33 ± 0.58 | 53.00 ± 1.73 | |
| 1000 | 93.00 ± 3.46 | 87.00 ± 1.73 | 78.00 ± 2.65 | 71.00 ± 1.00 | 67.67 ± 0.58 | 65.00 ± 1.3 | 64.00 ± 1.00 | 54.67 ± 0.58 | |
| 1500 | 97.43 ± 0.12 | 96.13 ± 1.15 | 89.33 ± 1.15 | 83.33 ± 1.53 | 75.67 ± 1.53 | 71.00 ± 1.00 | 66.33 ± 1.15 | 59.00 ± 1.73 | |
Data are presented as mean ± SD of three replications
the control; Fig. 7). The antifungal activity of amphotericin B on F. oxysporum was significantly greater than synthesized Ag-NPs by Fusarium oxysporum (P < 0.05; Tables 4 and 5).
Fig. 7.
Average percentage of growth inhibition of Fusarium at 50 nm (2 ppm) between days 2 to day 10
Table 4.
Effects of different concentrations of the produced AgNPs and amphotericin B on colony formation of F. oxysporum
| Days | Control | 200 ppm | 400 ppm | 600 ppm | |||
|---|---|---|---|---|---|---|---|
| AgNPs | Amphotericin B | AgNPs | Amphotericin B | AgNPs | Amphotericin B | ||
| 2 | 15.00 ± 0.00 | 1.07 ± 0.12 | 1.97 ± .06 | 1.00 ± 0.12 | 1.00 ± .00 | 0.50 ± 0.50 | |
| 3 | 19.67 ± 0.58 | 6.33 ± 0.58 | 2.80 ± .26 | 3.33 ± 0.58 | 2.00 ± .00 | 1.53 ± 0.58 | 1.15 ± 0.50 |
| 4 | 42.33 ± 1.53 | 14.67 ± 0.58 | 3.90 ± .10 | 10.33 ± 0.58 | 3.00 ± .00 | 7.67 ± 1.15 | 1.58 ± 0.00 |
| 5 | 53.33 ± 1.53 | 26.00 ± 1.00 | 4.83 ± .29 | 19.67 ± 0.58 | 3.93 ± .12 | 15.33 ± 0.58 | 2.30 ± 0.00 |
| 6 | 68.33 ± 1.53 | 31.67 ± 1.53 | 6.83 ± .29 | 24.67 ± 0.58 | 4.83 ± .29 | 24.00 ± 1.00 | 3.58 ± 0.50 |
| 7 | 79.67 ± 0.58 | 40.33 ± 0.58 | 8.87 ± .23 | 31.33 ± 1.53 | 5.77 ± .40 | 31.33 ± 1.15 | 4.80 ± 0.00 |
| 8 | 89.00 ± 1.73 | 49.00 ± 1.00 | 10.83 ± .29 | 39.00 ± 1.00 | 6.83 ± .29 | 38.67 ± 1.53 | 5.50 ± 0.50 |
| 9 | 99.00 ± 1.00 | 54.33 ± 1.15 | 14.50 ± .87 | 46.33 ± 1.53 | 7.93 ± .12 | 41.33 ± 1.53 | 6.70 ± 0.00 |
| 10 | 98.33 ± 2.08 | 60.67 ± 1.15 | 16.67 ± 1.53 | 54.33 ± 1.53 | 9.67 ± .58 | 51.00 ± 1.73 | 7.77 ± 0.00 |
Data are presented as mean ± SD of three replications
Table 5.
Effects of different concentrations of the synthesized AgNPs and amphotericin B on mean growth inhibition of F. oxysporum, as a percent of the negative control
| Days | 200 ppm | 400 ppm | 600 ppm | |||
|---|---|---|---|---|---|---|
| AgNPs | Amphotericin B | AgNPs | Amphotericin B | AgNPs | Amphotericin B | |
| 2 | 74.33 ± 1.15 | 86.43 ± 0.75 | 74.33 ± 1.15 | 93.30 ± 0.00 | 96.60 ± 0.00 | |
| 3 | 67.67 ± 2.52 | 85.73 ± .1.54 | 83.00 ± 2.65 | 89.80 ± 0.35 | 93.00 ± 2.65 | 94.90 ± 0.17 |
| 4 | 64.67 ± 1.53 | 90.73 ± 0..35 | 75.33 ± 0.58 | 92.83 ± 0.25 | 81.67 ± 3.21 | 96.40 ± 0.10 |
| 5 | 50.33 ± 0.58 | 91.00 ± .0.75 | 62.67 ± 0.58 | 92.33 ± 0.35 | 70.67 ± 1.53 | 96.23 ± 0.12 |
| 6 | 53.33 ± 3.21 | 89.73 ± .0.64 | 63.33 ± 1.15 | 92.87 ± 0.31 | 64.33 ± 0.58 | 95.57 ± 0.12 |
| 7 | 49.33 ± 1.15 | 88.33 ± .0.58 | 60.33 ± 2.08 | 92.60 ± 0.66 | 61.33 ± 1.15 | 94.97 ± 0.06 |
| 8 | 44.33 ± 2.08 | 87.53 ± .0.68 | 55.67 ± 1.15 | 91.27 ± 1.18 | 56.00 ± 2.00 | 94.13 ± 0.23 |
| 9 | 45.67 ± 1.15 | 85.33 ± .0.76 | 53.67 ± 1.53 | 91.97 ± 0.06 | 58.67 ± 1.53 | 93.90 ± 0.10 |
| 10 | 39.33 ± 1.15 | 82.67 ± .1.53 | 45.67 ± 1.15 | 90.13 ± 0.42 | 49.00 ± 1.73 | 92.80 ± 0.17 |
Data are presented as mean ± SD of three replications
Results of Inhibitory Effects of Silver Nanoparticles on Aspergillus Fumigatus
Fungul colony diameter was measured by culturing fungus at different concentrations of the synthesized NPs. Then, the growth inhibition percentage was calculated. The highest percentage of growth inhibition of NPs produced on Aspergillus fumigatus at 800 ppm was approximately 88%. It is observed that the synthesized NPs have a significant inhibitory effect on the growth inhibition of Aspergillus fumigatus (Tables 6 and 7). The antifungal activity of amphotericin B on A. fumigatus was significantly greater than synthesized Ag-NPs by Fusarium oxysporum (P < 0.05; Tables 8 and 9).
Table 6.
Growth of A. fumigatus (colony diameter, mm) on SDA medium with different concentrations of the synthesized AgNPs for 10 days
| Concentrations (ppm) | Days | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
| 0 (control) | 3.33 ± 0.58 | 8.33 ± 0.58 | 20.67 ± 1.15 | 28.00 ± 1.00 | 32.00 ± 2.00 | 38.00 ± 2.00 | 45.00 ± 2.00 | 58.67 ± 1.15 | |
| 50 | 3.67 ± 0.58 | 8.33 ± 0.58 | 12.00 ± 1.00 | 15.67 ± 0.58 | 19.67 ± 0.58 | 22.67 ± 0.58 | 26.00 ± 1.00 | 28.67 ± 0.58 | |
| 100 | 3.33 ± 0.58 | 6.33 ± 0.58 | 10.00 ± 1.00 | 15.33 ± 0.58 | 20.33 ± 1.53 | 23.67 ± 1.53 | 27.00 ± 2.00 | 31.00 ± 1.00 | |
| 150 | 2.33 ± 0.58 | 3.50 ± 0.58 | 4.33 ± 0.58 | 5.67 ± 0.76 | 6.33 ± 0.58 | 7.33 ± 0.58 | 10.67 ± 0.58 | 12.67 ± 0.58 | |
| 200 | 1.00 ± 0.00 | 2.33 ± 0.58 | 3.67 ± 0.58 | 4.13 ± 0.55 | 4.83 ± 0.58 | 7.33 ± 1.00 | 10.67 ± 1.15 | 12.67 ± 0.58 | |
| 400 | 1.00 ± 0.00 | 2.50 ± 0.50 | 4.00 ± 1.00 | 5.00 ± 1.00 | 5.67 ± 1.15 | 7.67 ± 1.15 | 8.67 ± 0.58 | 10.67 ± 0.58 | |
| 600 | 1.00 ± 0.00 | 2.17 ± 0.29 | 3.00 ± 0.58 | 3.67 ± 1.00 | 5.33 ± 0.58 | 5.67 ± 0.58 | 7.33 ± 0.58 | 11.00 ± 1.00 | |
| 800 | 1.33 ± .58 | 2.50 ± 0.50 | 2.68 ± 0.58 | 4.33 ± 0.58 | 5.67 ± 0.58 | 7.00 ± 1.00 | |||
| 1000 | 1.33 ± 0.58 | 2.67 ± 1.15 | 3.83 ± 0.58 | 4.17 ± 0.58 | 5.17 ± 1.15 | 7.00 ± 1.00 | 9.00 ± 0.58 | ||
| 1500 | 2.33 ± 0.58 | 3.50 ± 0.58 | 5.67 ± 0.58 | 6.67 ± 1.00 | 9.00 ± 1.00 | 11.33 ± 1.15 | 17.00 ± 1.15 | 21.67 ± 1.53 | |
Data are presented as mean ± SD of three replications
Table 7.
Mean growth inhibition of A. fumigatus (colony diameter, mm) on SDA medium with different concentrations of the produced AgNPs for 10 days, as a percent of the negative control
| Concentrations (ppm) | Days | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
| 50 | 42.33 ± 2.52 | 50.67 ± 3.51 | 38.33 ± 2.89 | 43.33 ± 3.06 | 38.33 ± 2.65 | 40.00 ± 3.46 | 41.67 ± 4.04 | 51.00 ± 1.73 | |
| 100 | 35.67 ± 1.15 | 24.00 ± 1.73 | 51.67 ± 2.89 | 44.67 ± 4.16 | 36.00 ± 1.00 | 37.00 ± 1.00 | 39.33 ± 3.79 | 46.67 ± 1.15 | |
| 150 | 27.67 ± 4.62 | 58.00 ± 3.61 | 79.00 ± 1.73 | 80.67 ± 1.53 | 79.67 ± 3.23 | 80.00 ± 2.65 | 75.66 ± 1.53 | 78.00 ± 1.73 | |
| 200 | 69.00 ± 5.20 | 72.00 ± 5.9 | 79.00 ± 1.73 | 84.67 ± 2.08 | 84.33 ± 3.06 | 80.00 ± 2.00 | 78.00 ± 1.00 | 79.33 ± 1.53 | |
| 400 | 69.00 ± 5.2 | 69.67 ± 4.73 | 80.33 ± 5.51 | 81.67 ± 3.06 | 81.77 ± 3.07 | 79.33 ± 2.07 | 80.33 ± 1.53 | 81.33 ± 0.58 | |
| 600 | 75.00 ± 0.00 | 74.00 ± 1.73 | 85.33 ± 0.58 | 86.67 ± 2.08 | 83.00 ± 1.00 | 84.67 ± 1.53 | 83.33 ± 1.53 | 81.00 ± 1.00 | |
| 800 | 93.33 ± 2.89 | 91.00 ± 2.65 | 91.33 ± 1.53 | 88.00 ± 1.00 | 87.00 ± 1.00 | 87.67 ± 2.08 | |||
| 1000 | 83.67 ± 3.73 | 86.67 ± 2.65 | 85.67 ± 2.89 | 86.67 ± 2.58 | 85.67 ± 1.53 | 84.00 ± 1.00 | 84.33 ± 2.58 | ||
| 1500 | 30.33 ± 3.12 | 57.67 ± 3.79 | 72.33 ± 4.04 | 75.67 ± 2.08 | 72.00 ± 2.65 | 70.00 ± 1.00 | 61.67 ± 1.53 | 62.33 ± 1.73 | |
Data are presented as mean ± SD of three replications
Table 8.
Effects of different concentrations of the produced AgNPs and amphotericin B on colony formation of A. fumigatus
| Days | Control | 200 ppm | 400 ppm | 600 ppm | |||
|---|---|---|---|---|---|---|---|
| AgNPs | Amphotericin B | AgNPs | Amphotericin B | AgNPs | Amphotericin B | ||
| 2 | 3.33 ± 0.58 | 2.67 ± 0.29 | 2.00 ± 0.00 | 1.00 ± 0.50 | |||
| 3 | 8.33 ± 0.58 | 1.00 ± 00 | 3.80 ± 0.26 | 1.00 ± 0.00 | 3.00 ± 0.00 | 1.00 ± 0.00 | 2.00 ± 0.50 |
| 4 | 20.67 ± 1.15 | 2.33 ± 0.58 | 4.90 ± 0.10 | 2.50 ± 0.50 | 4.04 ± 0.60 | 2.17 ± 1.15 | 3.00 ± 0.00 |
| 5 | 27.67 ± 0.58 | 3.67 ± 0.58 | 5.83 ± 0.29 | 4.00 ± 1.00 | 5.10 ± 0.17 | 3.00 ± 0.58 | 4.00 ± 0.00 |
| 6 | 31.00 ± 2.00 | 4.13 ± 1.53 | 7.83 ± 0.29 | 5.00 ± 1.00 | 6.17 ± 0.29 | 3.67 ± 1.00 | 4.93 ± 0.50 |
| 7 | 38.00 ± 2.00 | 4.83 ± 0.76 | 10.87 ± 0.23 | 5.67 ± 1.53 | 7.00 ± 0.40 | 5.33 ± 1.15 | 5.97 ± 0.58 |
| 8 | 44.33 ± 2.31 | 7.33 ± 0.58 | 12.83 ± 0.29 | 7.67 ± 1.00 | 8.00 ± 0.29 | 5.67 ± 1.53 | 6.93 ± 0.50 |
| 9 | 54.33 ± 2.31 | 9.67 ± 1.15 | 19.50 ± 0.87 | 8.67 ± 1.53 | 9.03 ± 0.12 | 7.33 ± 1.53 | 8.07 ± 0.58 |
| 10 | 64.33 ± 2.31 | 12.00 ± 1.15 | 24.67 ± 1.53 | 10.67 ± 1.53 | 10.17 ± 0.29 | 11.00 ± 1.73 | 9.47 ± 0.55 |
Data are presented as mean ± SD of three replications
Table 9.
Effects of different concentrations of the synthesized AgNPs and amphotericin B on mean growth inhibition of A. fumigatus, as a percent of the negative control
| Days | 200 ppm | 400 ppm | 600 ppm | |||
|---|---|---|---|---|---|---|
| AgNPs | Amphotericin B | AgNPs | Amphotericin B | AgNPs | Amphotericin B | |
| 2 | 19.00 ± 5.20 | 38.67 ± 3.00 | 69.00 ± 3.20 | |||
| 3 | 69.00 ± 5.2 | 54.07 ± 3.52 | 69.00 ± 5.20 | 63.67 ± 2.02 | 75.00 ± 0.00 | 75.90 ± 1.56 |
| 4 | 72.00 ± 5.2 | 76.23 ± 0.87 | 69.67 ± 4.73 | 80.17 ± 0.76 | 74.00 ± 1.73 | 85.33 ± 0.58 |
| 5 | 79.33 ± 0.58 | 78.33 ± 1.53 | 80.33 ± 5.51 | 81.33 ± 0.58 | 85.33 ± 1.53 | 85.60 ± 0.46 |
| 6 | 84.67 ± 2.08 | 75.47 ± 0.81 | 81.67 ± 3.08 | 80.00 ± 1.73 | 86.67 ± 2.08 | 84.33 ± 0.58 |
| 7 | 84.33 ± 3.21 | 71.00 ± 1.00 | 80.67 ± 2.08 | 79.67 ± 1.53 | 83.00 ± 1.15 | 84.00 ± 0.58 |
| 8 | 80.00 ± 2.08 | 70.57 ± 1.25 | 79.33 ± 1.15 | 79.67 ± 1.18 | 84.67 ± 2.00 | 83.67 ± 1.15 |
| 9 | 78.00 ± 1.15 | 64.30 ± 1.59 | 80.33 ± 1.53 | 80.00 ± 0.58 | 83.33 ± 1.53 | 83.67 ± 1.15 |
| 10 | 79.33 ± 1.15 | 61.73 ± 1.55 | 81.33 ± 1.15 | 79.00 ± 0.42 | 81.00 ± 1.73 | 83.33 ± 0.58 |
Data are presented as mean ± SD of three replications
Discussion
In the last decade, the development of biological systems as an environmentally friendly way of forming metal nanoparticle has become an interesting and important scientific field. Many microorganisms including bacteria, yeasts, filamentous fungi, algae, and plants, have shown to be capable of producing different types of metal NPs such as silver, gold, palladium, and others [12]. F. oxysporum fungus can generate different extracellular NPs, such as NPs of gold, silver, and their alloys, titanium, and bismuth. Various studies revealed extracellular proteins released into the solution by F. oxysporum which propounded a possible efficiency in reduction of metals and production of NPs [13].
In a study gold NPs from Rhizopus oryza were produced. The production of these NPs was verified by UV–vis spectrophotometry, observing the color change of the reaction solution from yellow to purple and creation of a specific peak at 540 nm. XRD analysis of the resulting NPs proved that the synthesized particles were gold nanocrystals. Images obtained by transmission electron microscopy, showed that the Rhizopus oryza intracellularly and extracellularly synthesized gold NPs in spherical and triangular shapes [14]. In another study, silver and gold NPs using Chrysosporium tropicum was synthesized [15]. The researchers investigated the effective factors of NP production including temperature, pH, light intensity, and fungus biomass. They found that the alkaline pH of 9 and 11, temperature of 40 °C to 60 °C, the sunlight as the light source, and 6 g of biomass in 100 ml distilled water, were the most important factors in producing the highest amount of Ag-NPs by Fusarium oxysporum [16]. In a similar study Ag-NPs were produced from Penicillium chrysogenum. They used UV–vis spectrophotometer to verify that the presence of a peak at 420 nm was due to the presence of Ag-NPs. They also used SEM and atomic force microscopy (AFM) to determine the average diameter of NPs (DLS), zeta potential, and NPs scattering index (Polydispersity Index) [17].
Extracellular production in all cases has different shapes and sizes. The reduction of metal ions in this fungus is due to the NADH reductase and alteration in extracellular production. The reduction is mainly due to the correlation between the electron shuttle with the participation of NADH reductase. Therefore, the reduction of silver nitrate salt and the production of Ag-NPs are done in this fungus [18]. Based on the results, to get the best value of the fungus biomass, the volume of the fungus should be double the amount of salt, and this is important for an economically viable mass production. But, in the current research, we used an equal volume of fungus biomass and salt, due to less needed time. Also, the best concentration for synthesis was 2 mM salt as to produce more NPs in less time. The best pH was selected as pH = 9 and the best time was 24 h after adding the fungus biomass to the silver nitrate salt solution. Spectrophotometer analysis revealed a distinct peak at 420 nm. This was a solid validation for the accuracy of silver nanoparticle production. The particles were round and without aggregation. The size of Ag-NPs was evaluated by FESEM and showed to be from 25 to 100 nm (Fig. 3). XRD patterns of Miller indices of values 111, 200, 220, and 311 correspond to angles of 38.143°, 46.255°, 64.51°, and 77.011°, respectively, which verify the presence of Ag-NPs in the biosynthetic method. The existence of silver atoms was attested using energy-dispersive X-ray spectroscopy. The result of TEM also demonstrated that most of the particles were spherical and with both cumulative and dispersed distribution.
Ghojavand et al. for the first time successfully performed green synthesis of Ag-NPs in 15 min using a prepared aqueous Felty germander extract with 15 mM AgNO3 solution at 30 °C and pH of 6. The resulting Ag-NPs were spherical in shape with sizes in the range 10–100 nm. Furthermore, these particles were clearly dispersed with no aggregation and showed antifungal activity against F. oxysporum [19].
Leal and colleagues succeeded in synthesizing Ag-NPs from Aspergillus foetidus These particles had antifungal activity on Aspergillus niger, Aspergillus flavus, Aspergillus foetidus and Fusarium oxysporum. In addition, the researchers demonstrated that their antifungal activity is due to the inactivation of sulfhydryl groups in the fungal cell wall and the impairment of membrane-bound enzymes [20]. Asharani and colleagues studied the antiproliferative effects of Ag-NPs and suggested a mechanism of toxicity for them. Ag-NPs can interact with membrane proteins and activate the signaling pathways that inhibit cell proliferation. Ag-NPs can also enter the cell via infiltration or endocytosis and cause mitochondrial dysfunction, then by the production of reactive oxygen species (ROS), cause damage to proteins and nucleic acids inside the cell and ultimately inhibit the cell proliferation [21]. An important toxic mechanism for Ag-NPs in both ionic and nano-forms is the interaction with sulfur-containing macromolecules, such as proteins, which is a result of the strong influence of silver on sulfur [22]. In another study, Ag-NPs were extracellularly synthesized by Fusarium oxysporum. The synthesized Ag-NPs were 20–50 nm in diameter. The reduction of metal ions had occurred by both nitrate-dependent reductase and an extracellular process by the Quinone shuttle, and their high potential as antibacterial agents was observed [23].
The results of this research revealed that the synthesis of Ag-NPs of 25–100 nm with Fusarium oxysporum exhibit a significant antifungal effect on Fusarium and Aspergillus fungi. The percentage of growth inhibition was studied at various nanoparticle concentrations (i.e., 50, 100, 150, 200, 400, 800, and 1500 ppm) and showed a direct dependency on that; an increase in the concentration led to an increase in the growth inhibition. The maximum growth inhibition percentage of Ag-NPs on Fusarium was approximately 60% at 1500 ppm, and 88% on Aspergillus at 800 ppm. The antibacterial effect of NPs is mainly determined by their size, shape, and concentration. Smaller NPs potentially have higher percentages of interactions compared to larger particles and show higher toxicity against bacterial pathogens, since these are more likely to diffuse easily relative to the larger ones [24].
The fungicidal activity mechanism of Ag-NPs is still debatable, though it has been propounded that Ag-NPs hinder the budding process through the formation of pores on the fungal cell membrane that can result in cell death [25, 26]. In this context, it is suggested that the antibacterial activity of Ag-NPs might be mediated by the formation of free radicals which leads to membrane damage and the formation of pits on the surface of the bacterial cell membrane [27]. Moreover, the generation of free radicals may cause severe damage to the chemical structure of DNA and proteins [28].
Also, according to our results, the percentage of growth inhibition of Amphotericin B is greater than synthetic Ag-NPs. Perhaps the only possible disadvantage was the nanoparticle's different behavior in inhibiting the fungus during the different days compared to amphotericin, which can be explained as Amphotericin B is a chemical compound produced under controlled conditions while our NPs are biologically produced and various factors such as temperature and humidity can affect their production. Therefore, the produced NPs are not uniform and may have different effects. These NPs were produced from the native Fusarium fungus of Isfahan city for the first time, and such a significant antifungal activity was obtained. Silver nanoparticle synthesis using fungi is a clean, green, inexpensive, environmentally friendly, reliable, and safe method and it can also be used for various applications in real life.
Though nanotechnology has great potential in terms of profitability, it may have direct or indirect effects on soil and aquatic ecosystems. Determining the toxicity of nanomaterials is a fundamental issue associated with size, surface area, shape and morphology of NPs, and their increased surface activity. To evaluate the toxicity of NPs, it is necessary to know the absorption rate of NPs in the body which is affected by various factors such as the shape and surface charge of the NPs. The NPs are able to penetrate into cells and even into the nucleus causing changes in protein and DNA cells. In general, NPs will interact with blood, the immune system, and body cells and lead to` toxicity by affecting their biological activity. They alter the structure of the protein and destroy its function. They can also interfere with the system activity and then reduce or increase its performance, which would have adverse effects in both cases. The extent of these toxicities has been evaluated by laboratory and clinical studies and its results are used to safely design workplaces, consumables, and related wastes [29]. There is currently no evidence of the adverse effects on humans via the products made with nanosilver. In general, Ag-NPs have less impact on humans compared to all metal NPs, and their contamination and risk are less than chemical drugs [30]. However, Ag-NP products may cause silver to dissolve, Ag-NPs entering the environment. The accumulation of these particles would have adverse effects on the environment and ecosystem [31, 32].
Conclusion
In this study, Ag-NPs were successfully synthesized using Fusarium oxysporum and the synthesized NPs had desirable structural traits. They also effectively inhibited the growth of Fusarium oxysporum and Aspergillus fumigatus.
Footnotes
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References
- 1.Honary S, Barabadi H, Gharaei-Fathabad E, et al. Green synthesis of silver nanoparticles induced by the fungus Penicillium citrinum. Trop J Pharm Res. 2013 doi: 10.4314/tjpr.v12i1.2. [DOI] [Google Scholar]
- 2.Honary S, Gharaei-Fathabad E, Barabadi H, et al. Fungus-mediated synthesis of gold nanoparticles: a novel biological approach to nanoparticle synthesis. J Nanosci Nanotechnol. 2013 doi: 10.1166/jnn.2013.5989. [DOI] [PubMed] [Google Scholar]
- 3.Ahmad A, Mukherjee P, Senapati S, et al. Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium oxysporum. Colloids Surf B. 2003 doi: 10.1016/S0927-7765(02)00174-1. [DOI] [Google Scholar]
- 4.Mukherjee P, Ahmad A, Mandal D, et al. Fungus-mediated synthesis of silver nanoparticles and their immobilization in the mycelial matrix: a novel biological approach to nanoparticle synthesis. Nano Lett. 2013 doi: 10.1007/s12645-013-0038-3. [DOI] [PubMed] [Google Scholar]
- 5.Ahmad N, Sharma S, Alam MK, et al. Rapid synthesis of silver nanoparticles using dried medicinal plant of basil. Colloids Surf B. 2010 doi: 10.1016/j.colsurfb.2010.06.029. [DOI] [PubMed] [Google Scholar]
- 6.Franci G, Falanga A, Galdiero S, et al. Silver nanoparticles as potential antibacterial agents. Molecules. 2015 doi: 10.1016/j.colsurfb.2010.06.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Durán N, Durán M, de Jesus MB, et al. Silver nanoparticles: a new view on mechanistic aspects on antimicrobial activity. Nanomedicine. 2016 doi: 10.1016/j.nano.2015.11.016. [DOI] [PubMed] [Google Scholar]
- 8.Hang XF, Liu ZG, Shen W, et al. Silver nanoparticles: synthesis, characterization, properties, applications, and therapeutic approaches. Int J Mol Sci. 2016 doi: 10.3390/ijms17091534. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Tarazona A, Gómez JV, Mateo EM, et al. Antifungal effect of engineered silver nanoparticles on phytopathogenic and toxigenic Fusarium spp. and their impact on mycotoxin accumulation. Int J Food Microbiol. 2019 doi: 10.1016/j.ijfoodmicro.2019.108259. [DOI] [PubMed] [Google Scholar]
- 10.Alarcon EI, Griffith M. Udekwu KI Silver nanoparticle applications. Berlin: Springer; 2015. [Google Scholar]
- 11.Ishida K, Cipriano TF, Rocha GM, et al. Silver nanoparticle production by the fungus Fusarium oxysporum: nanoparticle characterisation and analysis of antifungal activity against pathogenic yeasts. Mem Inst Oswaldo Cruz. 2014 doi: 10.1590/0074-0276130269. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Ghosh P, Han G, De M, et al. Gold nanoparticles in delivery applications. Adv Drug Deliv Rev. 2008 doi: 10.1016/j.addr.2008.03.016. [DOI] [PubMed] [Google Scholar]
- 13.Naimi-Shamel N, Pourali P, Dolatabadi S. Green synthesis of gold nanoparticles using Fusarium oxysporum and antibacterial activity of its tetracycline conjugant. J Mycol Med. 2019 doi: 10.1016/j.mycmed.2019.01.005. [DOI] [PubMed] [Google Scholar]
- 14.Sheikhlou Z, Salouti M, Farahmandkia Z, et al. Intra-extra biosynthesis of gold nanoparticles by fungus Rhizopus oryza. J Adv Med Biomed Res. 2012;20:47–56. [Google Scholar]
- 15.Soni N, Prakash S. Efficacy of fungus mediated silver and gold nanoparticles against Aedes aegypti larvae. Parasitol Res. 2012 doi: 10.1007/s00436-011-2467-4. [DOI] [PubMed] [Google Scholar]
- 16.Birla SS, Gaikwad SC, Gade AK, et al. Rapid synthesis of silver nanoparticles from Fusarium oxysporum by optimizing physicocultural conditions. Sci World J. 2013 doi: 10.1155/2013/796018. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Barabadi H, Honary S (2014) Biological synthesis of silver nanoparticles using standard fungus of Penicillium chrysogenum. RJMS. http://rjms.iums.ac.ir/article-1-3284-en.html
- 18.Durán N, Marcato PD, De Souza GI, et al. Antibacterial effect of silver nanoparticles produced by fungal process on textile fabrics and their effluent treatment. J Biomed Nanotech. 2007 doi: 10.1166/jbn.2007.022. [DOI] [Google Scholar]
- 19.Ghojavand S, Madani M, Karimi J. Green synthesis, characterization and antifungal activity of silver nanoparticles using stems and flowers of felty germander. J Inorg Organomet Polym Mater. 2020;85:96–89. doi: 10.1007/s10904-020-01449-1. [DOI] [Google Scholar]
- 20.Leal SM, Jr, Roy S, Vareechon C, et al. Targeting iron acquisition blocks infection with the fungal pathogens Aspergillus fumigatus and Fusarium oxysporum. PLoS Pathog. 2013 doi: 10.1371/journal.ppat.1003436. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Asharani PV, Hande MP, Valiyaveettil S. Anti-proliferative activity of silver nanoparticles. BMC Cell Biol. 2009 doi: 10.1186/1471-2121-10-65. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Levard C, Mitra S, Yang T, et al. Effect of chloride on the dissolution rate of silver nanoparticles and toxicity to E. coli. Environ Sci Technol. 2013 doi: 10.1021/es400396f. [DOI] [PubMed] [Google Scholar]
- 23.Durán N, Marcato PD, Alves OL, et al. Esposito E, Mechanistic aspects of biosynthesis of silver nanoparticles by several Fusarium oxysporum strains. J Nanobiotechnology. 2005 doi: 10.1186/1477-3155-3-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Buszewski B, Railean-Plugaru V, Pomastowski P, et al. Antimicrobial activity of biosilver nanoparticles produced by a novel Streptacidiphilus durhamensis strain. J Microbiol Immun Infect. 2018 doi: 10.1016/j.jmii.2016.03.002. [DOI] [PubMed] [Google Scholar]
- 25.Jalal M, Ansari MA, Alzohairy MA, et al. Biosynthesis of silver nanoparticles from oropharyngeal Candida glabrata isolates and their antimicrobial activity against clinical strains of bacteria and fungi. Nanomaterials. 2018 doi: 10.3390/nano8080586. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Qais FA, Khan MS, Ahmad I, et al. Potential of nanoparticles in combating Candida infections. Lett Drug Design Discovery. 2019 doi: 10.2174/1570180815666181015145224. [DOI] [Google Scholar]
- 27.Paulkumar K, Gnanajobitha G, Vanaja M, et al. Piper nigrum leaf and stem assisted green synthesis of silver nanoparticles and evaluation of its antibacterial activity against agricultural plant pathogens. Sci World J. 2014 doi: 10.1155/2014/829894. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Marambio-Jones C, Hoek EM. A review of the antibacterial effects of silver nanomaterials and potential implications for human health and the environment. J Nanopart Res. 2010 doi: 10.1007/s11051-010-9900-y. [DOI] [Google Scholar]
- 29.Zhu Z, Rezhdo O, Perrone M, et al. Magnetite nanoparticles doped photoresist derived carbon as a suitable substratum for nerve cell culture. Colloids Surf B. 2013 doi: 10.1016/j.colsurfb.2012.07.045. [DOI] [PubMed] [Google Scholar]
- 30.Kumar A, Vemula PK, Ajayan PM, et al. Silver-nanoparticle-embedded antimicrobial paints based on vegetable oil. Nat Mater. 2008 doi: 10.1038/nmat2099. [DOI] [PubMed] [Google Scholar]
- 31.Geranio L, Heuberger M, Nowack B. The behavior of silver nanotextiles during washing. Environ Sci Technol. 2009 doi: 10.1021/es9018332. [DOI] [PubMed] [Google Scholar]
- 32.Gottschalk F, Sonderer T, Scholz RW, et al. Modeled environmental concentrations of engineered nanomaterials (TiO(2), ZnO, Ag, CNT, Fullerenes) for different regions. Environ Sci Technol. 2009 doi: 10.1021/es9015553. [DOI] [PubMed] [Google Scholar]