Analysis of molecular biodiversity in toxigenic fungi of agri-food interest

Filamentous fungi are well known for their ability to produce numerous secondary metabolites, such as antibiotics, pigments, and mycotoxins. Fungi belongingto the genera Aspergillus, Fusarium, and Penicillium are the most common fungi associated with the contamination of crop plants with mycotoxins, representing an important risk to human and animal health. Most fungal secondary metabolites are products of multi-step biochemical pathways that often begin with a terpene synthase, polyketide synthase or non-ribosomal peptide synthetase that undergo a series of enzyme-catalyzed modifications to form the mycotoxins, which is a significant concern for food and feed safety. The goal of this thesis was to investigate the distribution of genes/clusters related to potential mycotoxins biosynthetic capability within heterogeneous taxa occurring on cereals, like Aspergillus niger clade and the Fusarium incarnatum-equiseti species complex (FIESC). Despite their sporadic occurrence on cereals, little is known about their potential contribution in mycotoxins contamination of cereals. We first evaluated in maize samples, from USA and Italy, the species composition of Aspergillus sect. Nigri¸ to determine rates of 2 phylogenetic species, A. niger and A. welwitschiae, through housekeeping genes-based phylogeny. We also evaluated the related fumonisins biosynthetic capability, through assessment of the occurrence of fum8 gene, as part of FUM cluster, and evaluation in vitro of fumonisin B2 production. Phylogenetic analysis allowed us to characterize the Aspergillus population in maize, showing that A. niger was predominant among the USA isolates while A. tubingensis and A. welwitschiae occurred at higher frequencies in the Italian maize crops. Furthermore, a higher percentage of A. niger isolates produced FB2 compared to A. welwitschiae, suggesting a higher fumonisin contamination risk in the USA population. Finally, we demonstrated that presence of fum8 gene in the genome is not correlated with the production of fumonisin in vitro, confirming the ineffectiveness of fum8 presence as a predictor of FB2 production. We then evaluated the molecular biodiversity in FIESC, a heterogeneous and complex species, including a high number of phylogenetic species (30) with boundaries not completely elucidated within this complex. For the analysis we chose a polyphasic approach, including the whole genome sequencing. Housekeeping genes-based phylogeny allowed us to resolve the majority of isolates into four previously described phylogenetic species of FIESC, and identify a novel species, designated in this work as FIESC 31. Phylogenies based on nucleotide sequences of trichothecene biosynthetic genes and MALDI-TOF MS data were largely concordant with the housekeeping gene based species phylogeny. Chemical analysis revealed variability in trichothecenes production profiles among the different investigated phylogenetic species. Finally, in an effort to better understand the biology of this fungus, we performed genome sequencing of twelve FIESC strains revealing a marked variation in distribution of mycotoxin biosynthetic gene clusters among phylogenetic species. All these results contribute to an increased knowledge of secondary metabolites synthesis in toxigenic fungi. This offers a basis to develop rapid and reliable assays for fungal identification, hopefully applicable in “prevention” steps of crop protection programs, against mycotoxin contamination.