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The fungal community composition and structure of two ancient tetraploid wheat varieties, native to the Sicilian territory of Italy, Perciasacchi (winter wheat) and Tumminia (spring wheat) were investigated using High Throughput Sequencing (HTS). This showed a predominance of Ascomycetes and Basidiomycetes including Alternaria, Fusarium, Mycosphaerella, Filobasidium, Cystofilobasidium, Cryptococcus, Leucosporidium, Dioszegia, Puccinia, Sporobolomyces, Aureobasidium, Cladosporium, Holtermanniella and Gibberella. Principal Coordinates Analysis (PCoA) and Linear discriminant analysis Effect Size (LEfSe) showed that Aureobasidium, Leucosporidium and Puccinia differentiated between the two wheat varieties. In addition, the microbial association analysis suggested that some endophytic taxa play important roles within the wheat fungal community. Genera such as Cryptococcus and Cystofilobasidium were shown to have consistent antagonistic activity against Gibberella spp., while, Acremonium and a group of unidentified ascomycetes had mutual exclusion relationships with Puccinia. Since both Gibberella and Puccinia contain several economically important pathogens of wheat, the detected fungal interactions may indicate microbial-mediated resistance in these wheat varieties.