A rapid Multiwell Test to assay the effect of natural metabolites on growth and mycotoxin production of Aspergillus flavus

Plant and microorganisms can be natural sources of bioactive metabolites with antioxidant, antimicrobial, or anti-inflammatory effects. The use of plant or microbial bioactive molecules in the integrated management of crop diseases represent an innovative sustainable choice to counteract the contamination of mycotoxins produced by phytopathogenic fungi as Aspergillus flavus. Here we report a rapid and effective methodology, Multiwell Test, employed to understand the effect of natural metabolites on the A. flavus growth and aflatoxins (AF) production. Multiwell Test allows monitoring simultaneously the effect on A. flavus of several natural metabolites at numerous concentrations. Plate spectrophotometer/fluorimeter was used to monitor the fungal growth (from 1 to 3-day post inoculation) and AF biosynthesis (by fluorescence); at 7-day post inoculation AF were extracted directly from the wells and analyzed by LC coupled to mass spectrometry. Natural metabolites tested derive from fungi and plants. Fungal compounds were obtained from Trametes versicolor, Pleurotus eryngii and Schyzophyllum commune (Basidiomycota). Notably, T. versicolor produces a bioactive exo-polysaccharide: Tramesan efficient in blocking completely AF synthesis. Plant-based extracts and compounds were obtained from officinal plants as Heracleum persicum, Crocus sativus, Peganum harmala, Trachyspermum, ammi Rosmarinus officinalis, Anethum graveolens, Berberis vulgaris and Berberis thunbergii via an hydroalcoholic extraction and from Linaria purpurea via aqueous extraction and subsequent purification. The Multiwell Tests indicate that the bioactive molecules derived from fungal cultures and plants extracts do not significatively affect the growth of A. flavus but have a role on the aflatoxin production. In particular aflatoxins levels showed a significant decrease in presence of T. versicolor compounds and their production were completely inhibited in presence of H. persicum, P. harmala, T. ammi and L. purpurea plant extracts. AF inhibition occur at transcriptional level for most compounds. Further investigations with machine learning algorithms will be undertaken in a quantitative composition-activity relationship (QCAR) fashion to shed light on the role of the extract components and pure compounds in modulating the aflatoxin levels. Our findings encouraging the use of natural bioactive compounds to manage the mycotoxin contamination in pre- and post-harvest disease of agricultural crops.