Antifungal activity of silver nanoparticles during in-vitro culture of Stevia rebaudiana Bertoni

Abstract

30Contamination by fungi and bacteria during the in-vitro propagation of plants leads to considerable losses of biological 31 material and precludes phytosanitary certification. The anti-microbial effect of silver nanoparticles (AgNPs) may be 32 an alternative for the eradication of in-vitro contaminants. This study evaluated the microbicidal activity of AgNPs on 33 a recurrent fungus during the micropropagation of stevia (Stevia rebaudiana Bertoni). First, the fungus was isolated 34 and identified at a molecular level by the sequencing and analysis of the ITS4/ITS5 rDNA region. The results of the 35 phylogenetic analysis of various fungi species showed that the strain under study (16-166-H) belongs to the genus 36 Sordaria and is 86.74% similar to S. tomento-alba (strain CBS 260.78). Subsequently, the inhibition of the growth of 37 S. tomento-alba was tested under different concentrations of AgNPs (0, 25, 50, 100, and 200 mg L -1 ), observing that 38 50 and 100 mg L -1 achieve ca. 50% growth inhibition (IC 50 ), while 200 mg L -1 produces a drastic inhibition. On the 39 other hand, the shape and size of AgNPs was examined using transmission electron microscopy (TEM), and the 40 transport and accumulation of AgNPs in S. tomento-alba cells were monitored through multiphoton microscopy. The 41 morphological and fluorescence analyses showed that AgNPs display different sizes, with larger nanoparticles retained 42 in fungal cell walls while smaller AgNPs penetrate into fungal cells. Probably, apoplastic and symplastic mechanisms 43 involved in the accumulation and transport of AgNPs affect the metabolic processes of the fungus, thus inhibiting its 44 growth. These results suggest that AgNPs possess antifungal activity and can be used in the eradication of contaminants 45 during the in-vitro culture of plant species.46 47