Molecular Identification and Phylogenetic Analysis of Fusarium spp. Associated with Barley Grains Using DNA Barcoding in Bulgaria

This study examined the genetic relationships and distribution frequencies of Fusarium species iso¬lated from barley in Bulgaria, using DNA barcoding and phylogenetic analysis. Molecular identification targeted ITS and β-tubulin regions, revealing seven Fusarium species. With 99.2–100% sequence similarity to reference Fusarium strains in GenBank, validating the accuracy of ITS and β-tubulin loci for distinguish¬ing species. Among the identified species, F. sporotrichioides was the most frequent, with a prevalence of 46.1%. F. proliferatum followed, showing a lower but notable frequency of 15.4%, while F. poae and F. verticillioides each appeared in 8% of the isolates. Other species, including F. tricinctum and F. avenaceum, were less prevalent. Phylogenetic analyses, constructed using the Neighbor-Joining method, revealed geo¬graphic clustering trends. Bulgarian F. sporotrichioides isolates clustered closely with Chinese strains in the ITS phylogeny, suggesting a homogeneous population with ties to Asian origins. In contrast, F. avenaceum showed genetic diversity, clustering with both Chinese and Polish strains. F. brachygibbosum, a novel find in Bulgarian barley, aligned genetically with Russian strains, indicating potential cross-regional exchange. The β-tubulin-based phylogeny presented some distinctions from ITS, as F. sporotrichioides clustered with Russian and Finnish strains, underscoring the value of multi-locus barcoding for comprehensive phyloge¬netic insights. These findings highlight the diverse origins of Fusarium species in Bulgarian barley, which carry implications for disease management in the context of evolving climate conditions.