Antifungal activity of silver nanoparticles during in-vitro culture of Stevia rebaudiana Bertoni
Contamination by fungi and bacteria during the in-vitro propagation of plants leads to considerable losses of biological material and precludes phytosanitary certification. The anti-microbial effect of silver nanoparticles (AgNPs) may be an alternative for the eradication of in-vitro contaminants. This study evaluated the microbicidal activity of AgNPs on a recurrent fungus during the micropropagation of stevia (Stevia rebaudiana Bertoni). First, the fungus was isolated and identified at a molecular level by the sequencing and analysis of the ITS4/ITS5 rDNA region. The results of the phylogenetic analysis of various fungi species showed that the strain under study (16-166-H) belongs to the genus Sordaria and is 86.74% similar to S. tomento-alba (strain CBS 260.78). Subsequently, the inhibition of the growth of S. tomento-alba was tested under different concentrations of AgNPs (0, 25, 50, 100, and 200 mg L-1), observing that 50 and 100 mg L-1 achieve ca. 50% growth inhibition (IC50), while 200 mg L-1 produces a drastic inhibition. On the other hand, the shape and size of AgNPs was examined using transmission electron microscopy (TEM), and the transport and accumulation of AgNPs in S. tomento-alba cells were monitored through multiphoton microscopy. The morphological and fluorescence analyses showed that AgNPs display different sizes, with larger nanoparticles retained in fungal cell walls while smaller AgNPs penetrate into fungal cells. Probably, apoplastic and symplastic mechanisms involved in the accumulation and transport of AgNPs affect the metabolic processes of the fungus, thus inhibiting its growth. These results suggest that AgNPs possess antifungal activity and can be used in the eradication of contaminants during the in-vitro culture of plant species.