Molecular Identification and Phylogenetic Analysis of Fusarium spp. Associated with Triticum aestivum L. Based on DNA Barcoding

Fusarium spp. are active producers of mycotoxins that enter the food chain and pose risks to human health. Identifying pathogenic agents is a key step in developing disease management strategies. For the first time in Bulgaria, we identified eight Fusarium species in wheat, harvest 2024–2025, through the application of DNA barcoding. For a genetic marker and construction of phylogenetic tree, the protein-coding gene β-tub was chosen. Among 26 identified isolates, F. sporotrichioides (42.3%) dominated, followed by F. proliferatum 23.1%), F. avenaceum (7.7%), F. armeniacum (7.7%), аnd F. poae (7.7%). F. tricinctum (3.8%), F. oxysporum (3.8%), and F. equiaseti (3.9%) were weakly expressed. Phylogenetic analysis classified the isolates into five species complexes: FSAMSC, FFSC, FTSC, FIESC, and FOSC and highlighted the genetic distances between them. Molecular genetic analysis showed that 84.6% of the wheat samples contained only one species of Fusarium, and in 15.4% the co-presence of two species was established. The largest share was in samples with a low infestation of 2–4%, which represented 35% (n = 32) of all positives. No statistically significant difference was found between varieties and contamination level, but a statistically significant positive correlation was demonstrated by the preceding crop (rapeseed, sunflower, and maize).