Eight novel diagnostic markers differentiate lineages of the highly invasive myrtle rust pathogen .

is the causal agent of myrtle rust in over 480 species within the family Myrtaceae. Lineages of are structured by their hosts in the native range, and some have success in infecting newly encountered hosts. For example, the pandemic biotype has spread beyond South America, and proliferation of other lineages is an additional risk to biodiversity and industries. Efforts to manage incursions, including lineage differentiation, relies on variable microsatellite markers. Testing these markers is time-consuming, complex, and requires reference material that is not always readily available. We designed a novel diagnostic approach targeting eight selectively chosen loci including the fungal mating-type (homeodomain) transcription factor locus. The locus ( and ) is highly polymorphic, facilitating clear biological predictions about its inheritance from founding populations. To be considered as potentially derived from the same lineage, all four alleles must be identical. If all four alleles are identical six additional markers can further differentiate lineage identity. Our lineage diagnostics relies on PCR amplification of eight loci in different genotypes of followed by amplicon sequencing using Oxford Nanopore Technologies (ONT) and comparative analysis. The lineage-specific assay was validated on four isolates with existing genomes, uncharacterized isolates, and directly from infected leaf material. We reconstructed alleles from amplicons and confirmed their sequence identity relative to their reference. Genealogies of alleles confirmed the variations at the loci among lineages/isolates. Our study establishes a robust diagnostic tool for differentiating known lineages of based on biological predictions and available nucleotide sequences. This tool is suited to detecting the origin of new pathogen incursions.