Silver nanoparticle production by the fungus Fusarium oxysporum: nanoparticle characterisation and analysis of antifungal activity against pathogenic yeasts

Abstract

The microbial synthesis of nanoparticles is a green chemistry approach that combines nanotechnology and microbial biotechnology. The aim of this study was to obtain silver nanoparticles (SNPs) using aqueous extract from the filamentous fungus Fusarium oxysporum as an alternative to chemical procedures and to evaluate its antifungal activity. SNPs production increased in a concentration-dependent way up to 1 mM silver nitrate until 30 days of reaction. Monodispersed and spherical SNPs were predominantly produced. After 60 days, it was possible to observe degenerated SNPs with in additional needle morphology. The SNPs showed a high antifungal activity against Candida and Cryptococcus , with minimum inhibitory concentration values ≤ 1.68 µg/mL for both genera. Morphological alterations of Cryptococcus neoformans treated with SNPs were observed such as disruption of the cell wall and cytoplasmic membrane and lost of the cytoplasm content. This work revealed that SNPs can be easily produced by F. oxysporum aqueous extracts and may be a feasible, low-cost, environmentally friendly method for generating stable and uniformly sized SNPs. Finally, we have demonstrated that these SNPs are active against pathogenic fungi, such as Candida and Cryptococcus.

Fig. 1. absorption spectrum from 20-600 nm of silver nanoparticles produced by aqueous extracts of Fusarium oxysporum and several concentrations of silver nitrate (AgNO ₃) solutions at different times (0-60 days) after initiation of the reaction. A: 0.5 mM AgNO ₃; B: 1.0 mM AgNO ₃; C: 1.5 mM AgNO ₃ ; D: 2.0 mM AgNO ₃.

Fig. 2. extracellular silver nanoparticles (SNPs) production by silver nitrate solutions mixed with aqueous extract from Fusarium oxysporum over time (up to 60 days). The absorption of the SNPs was measured at 440 nm. *: p < 0.05; **: p < 0.01.

Fig. 3. transmission electron microscopy characterisation of silver nanoparticles produced during different reaction periods. A: five days; B: 30 days; C: 60 days. Bars = 200 nm; D: the presence of silver atoms (black arrow) was confirmed by energy-dispersive X-ray spectroscopy; Ag: silver; C: carbon; Cu: copper; O: oxygen; P: phosphorus; S: sulphur; Si: silicon.

Fig. 4. microscopic characterisation of silver nanoparticles (SNPs) produced by aqueous extracts of Fusarium oxysporum and 1.0 mM silver nitrate over five days. A: the presence of metallic particles was detected by scanning electron microscopy; B: the SNPS size and depth are indicated by the colour scale at the side of the atomic force microscopy micrograph.

Fig. 5. morphological alterations of Cryptococcus neoformans treated with sub-inhibitory concentrations of silver nanoparticles (SNPs) (0.21 µg/mL) for 72 h at 35ºC. The untreated yeast exhibit a compact cell wall (CW), continuous cytoplasmic membrane (cm), homogeneous and electron-dense cytoplasm and a polysaccharide capsule (c) surrounding the cell (A, B). By contrast, yeasts treated with SNPs had a disrupted cytoplasmic membrane and CW (asterisk) and increased cell wall thickness (CW) (C, D). The SNPs appear to be retained in the polysaccharide capsule (F, black arrowhead).