Fungal mediated extracellular biosynthesis of gold and silver nanoparticles and their effectiveness against seed-borne pathogens

In recent years, remarkable progress has been made in developing nanotechnology, which has led to the fast growth of commercial applications in diverse fields including agriculture (e.g. nanofertilizers, nanopesticides, nanoherbicides, nanoparticles mediated delivery of genetic material for crop protection and nanosensors for pathogen detection and soil monitoring). Metallic nanoparticles (MeNPs) are one of the building blocks of nanotechnology; they have a variety of applications due to their unique properties. The synthesis of MeNPs can be carried out by using traditional chemical and physical methods, but these are expensive and harmful with a low production rate. A new green chemistry approach that interconnects nanotechnology and biotechnology is the extracellular biosynthesis of MeNPs mediated by fungi, which are preferred to other biological systems for a large-scale production. This approach is rapid, cost effective, and eco-friendly. The aim of this work was to induce the mycosynthesis of silver and gold nanoparticles (AgNPs, AuNPs) and to evaluate their antimicrobial potential. For this purpose, eight fungi (Trichoderma longibrachiatum, T. harzianum, T. asperelloides, T. hamatum, T. paraviridescens, T. afroharzianum, Arthrinium phaeospermum and Pestalotiopsis sp.) isolated from deep sterilized plant and seed tissues were used. Cell-free fungal extracts were challenged with AgNO3 (2 mM) and HAuCl4 (1 mM) solutions. The resulting MeNPs were characterized by means of spectroscopic UV-Vis analysis and for two fungi TEM, EDAX and FTIR analyses were also performed. The antimicrobial activity of bioproduced MeNPs was tested by a microdilution assay in 96-well microtiter plates against five seed-borne pathogens: Colletotrichum lupini, Fusarium oxysporum f.sp. basilici, Botrytis cinerea, Xanthomonas campestris pv campestris and Pseudomonas syringae pv tomato. The AgNPs were very effective towards fungi. The best ones showed a growth inhibition percentage ranging from 87.0% to 99.4% for C. lupini, 91.2% to 100% for F. oxysporum f.sp. basilici and 91.2% to 100% for B. cinerea. Instead, the AuNPS showed limited inhibitory activity vs fungi that did not exceed 56%. The AgNPs were very effective also towards bacteria. The best ones showed a growth inhibition percentage ranging from 96% to 100% for X. campestris pv campestris and 83.4% to 100% for P. syringae pv tomato. Unlike fungi, bacteria were more sensitive to AuNPs showing growth inhibition percentage ranging from 67.8% to 81.8 % for X. campestris pv campestris and 63.7% to 92.1% for P. syringae pv tomato. Among fungi, the most effective against fungal pathogens was T. longibrachiatum while the most effective against bacteria was A. phaeospermum. Among seed-borne fungi, the most sensitive to the treatments was B. cinerea, whereas C. lupini seemed to be the less sensitive. Regarding the treated bacteria P. syringae pv tomato was the most sensitive to the treatments. The effectiveness of AgNPs produced by T. longibrachiatum and iprodione (conventional fungicide) was evaluated against C. lupini. Artificially infected lupine seeds were treated using two seed dressing protocols: a 2% Methocel A4M solution containing drugs or the drug solutions alone. Results from these preliminary experiments showed a lower inhibition activity of both drugs compared to that obtained with in vitro assays. The promising results obtained open up opportunities for further research on effective and ecofriendly solutions for the control of seed-borne diseases using mycosynthesized nanoparticles