Mating-compatibility genes employed as diagnostic markers to identify novel incursions of the myrtle rust pathogenAustropuccinia psidii

Abstract

ABSTRACTAustropuccinia psidiiis the causal agent of myrtle rust in over 480 species within the family Myrtaceae. Lineages ofA. psidiiare structured by host in its native range, and some have success on new-encounter 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 manageA. psidiiincursions, including lineage differentiation, relies on variable microsatellite markers. Testing these markers is time-consuming and complex, particularly on a large scale. We designed a novel diagnostic approach targeting the fungal mating-typeHD(homeodomain) transcription factor locus to address these limitations. TheHDlocus (bW1/2-HD1 andbE1/2-HD2)is highly polymorphic, facilitating clear biological predictions about its inheritance from founding populations. To be considered the same lineage, all fourHDalleles must be identical. Our lineage diagnostics relies on PCR amplification of theHDlocus in different genotypes ofA. psidiifollowed 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 reconstructedHDalleles from amplicons and confirmed their sequence identity relative to their reference. Genealogies usingHDalleles confirmed the variations at theHDloci among lineages/isolates. Our study establishes a robust diagnostic tool, for differentiating known lineages ofA. psidiibased biological predictions. This tool holds promise for detecting new pathogen incursions and can be refined for broader applications, including air-sample detection and mixed-isolate infections.