New informative molecular markers for Alternaria sections Infectoriae and Pseudoalternaria reveal the diversity of lineages infecting wheat and tomato

Fungi in Alternaria sections Infectoriae and Pseudoalternaria are commonly associated with commercially important crops, yet their biosystematics remains difficult to resolve because standard molecular markers often lack sufficient phylogenetic resolution. In this study, we used candidate loci identified in previous phylogenomic analyses to develop four new molecular markers (SIP-03, SIP-04, SIP-06, and SIP-08) that were specifically designed to be highly informative for these two sections. The performance of these new markers was evaluated using 237 strains collected primarily from wheat in Canada and tomatoes in the Baltic states, representing the largest data set analyzed to date. The four markers showed high amplification success and contained strong phylogenetic signal. Combined phylogenetic analyses recovered all of the previously recognized lineages within sections Infectoriae (Inf1, Inf3, Inf3) and Pseudoalternaria (Pse1, Pse2) and revealed a novel lineage, designated Inf4, associated with wheat in western Canada. Concordance of lineage assignment was high, with 94.1% of strains being placed consistently across all four markers and an overall marker conflict frequency of less than 3%. Our expanded sampling identified some lineage-specific patterns in regard to host association and geographic distribution. For example, the Inf1 lineage showed high molecular diversity and was dominant among tomato-associated strains from Baltic Europe, whereas Inf2 and Pse2 were the primary lineages infecting western Canadian wheat. Our study provides robust new tools for identification and classification of taxa within sections Infectoriae and Pseudoalternaria, refines their phylogenetic framework, and facilitates future studies on the pathology, epidemiology, and mycotoxin production of these agriculturally important fungi.