Chromosome-level genome assemblies for the latent pine pathogen, Diplodia sapinea, reveal two accessory chromosomes with distinct genomic features and evolutionary dynamics.

Diplodia sapinea (Dothideomycetes) is a latent fungal pathogen with a global distribution that predominantly infects Pinus species. The impact of the fungus is increasing due to climate-driven range expansion and thus wide-scale disease outbreaks are occurring. With the aim of developing high-quality genome resources, we generated chromosome-level genome assemblies for 3 D. sapinea isolates and low-coverage Illumina genome data for 6 additional isolates. By comparing these genome assemblies, we identified 14 core chromosomes and 2 accessory chromosomes (ACs) in the pathogen. These 2 ACs encode 80 and 147 proteins, respectively, while 11,374 to 11,601 genes were identified in the core chromosomes. Both ACs had lower gene density and higher proportions of transposable elements compared to the core chromosomes. Sequence analysis indicated that genes on the ACs displayed more sequence variation compared to those on the core chromosomes, suggesting they serve as evolutionary hotspots in the species. Sequence homology analyses suggested that the ACs were possibly acquired horizontally, probably from a species in the Dothideomycetes. We designed PCR-based assays to detect the presence of ACs and applied these on a set of 37 isolates from 14 countries. One of the ACs was detected in 33 isolates from 13 countries, while the other AC was absent in all isolates tested. Pathogenicity trials on Pinus patula seedlings showed no correlation between the presence of ACs and isolate aggressiveness. The high-quality genomes provided here offer important resources for future research on this globally important pathogen, including the biological roles of the ACs.