New molecular markers for distinguishing the main phylogenetic lineages within Alternaria section Alternaria

Abstract

Several members of Alternaria section Alternaria are economically important plant-pathogenic fungi that cause disease on a wide range of host types and plant tissues. The production of Alternaria-derived mycotoxins can lead to significant post-harvest losses due to contamination of agricultural products. Multiple Alternaria species are listed as regulated organisms, which are monitored by international plant protection programmes. These taxa often share high levels of both morphological and phylogenetic similarity, and the establishment of molecular markers that are able to distinguish among them unambiguously has proven to be quite challenging. Previously, we examined fine-scale genome-wide phylogenetic patterns and proposed a list of candidate genes for development into informative markers that are diagnostic for the main section Alternaria lineages. Here, we design primer sets and sequence three new markers (ASA-05, ASA-10, ASA-19) in order to evaluate their diagnostic performance. Sequence data for 49 new independent test taxa were combined with existing data for 38 taxa, and phylogenetic analyses revealed that, in general, the new molecular markers consistently classified section Alternaria strains into one of the main lineages. A discrepancy among the three markers for lineage assignment was observed only for one strain. We also sequenced a commonly used marker in molecular phylogenetics of fungi (rpb2, RNA polymerase II second largest subunit) and found it was outperformed by all three of the new markers. We suggest that an ASA marker presented here could form the basis of a convenient one-locus test for rapid and routine diagnostic screening of unknown section Alternaria strains.