Advanced rDNA-Based Detection of Wheat Pathogens in Grain Samples Using Next-Generation Sequencing (NGS)

High-throughput sequencing (HTS) has revolutionized phytopathology by overcoming many limitations of traditional diagnostic methods, as it permits precise pathogen monitoring, identification, and control, with ribosomal DNA (rDNA) regions serving as reliable markers for fungal classification. In this study, next-generation sequencing (NGS) was used, targeting the ITS1 and ITS2 regions to explore fungal diversity and pathogen presence in winter wheat grain samples and identifying 183 OTU sequences across 115 taxa. The ITS1 analysis yielded 249,743 reads, with <i>Fusarium</i> sp. (61%) as the dominant pathogenic taxon, followed by <i>Sporobolomyces</i> sp. (14%), <i>Cladosporium</i> sp. (3%), and other yeast-like or saprotrophic fungi, such as <i>Cryptoccocus</i> spp., <i>F. wieringae,</i> and <i>B. alba</i>. Sequencing of ITS1 also permitted the detection of <i>F. acuminatum</i> and the quarantine-regulated pathogens <i>T. caries</i> and <i>T. triticoides</i>. The ITS2 analysis produced 179,675 reads, with <i>F. culmorum</i> (47%) as the most abundant taxon, confirming significant grain contamination with this pathogen. Other frequently detected taxa included yeast-like fungi such as <i>C. tephrensis</i> (21%) and <i>V. victoriae</i> (13%), along with saprotrophic species like <i>S. roseus</i> and <i>Davidella</i> sp. ITS2 provided better resolution for the identification of <i>Fusarium</i> species by the detection of more pathogenic taxa associated with cereal diseases, including <i>F. culmorum,</i> as well as <i>F. cerealis</i>, <i>F. poae</i>, and <i>F. tricinctum</i>. The analysis revealed a diverse fungal community, including other pathogens such as <i>A. porri</i>, <i>B. cinerea</i>, and <i>C. herbarum</i>, as well as various non-pathogenic and saprotrophic fungal taxa. These findings underscore the complementary utility of ITS1 and ITS2 in profiling fungal diversity and detecting critical pathogens using HTS, highlighting the potential of these DNA regions for monitoring and managing cereal crop health.