Comparative Genomics and In Planta Transcriptomics Elucidate the Genetic Arsenal Used by Boxwood Blight Pathogen <i>Calonectria henricotiae</i>

Boxwood blight is a devastating fungal disease causing significant economic losses to the nursery industry and damaging established landscapes. The European pathogen, Calonectria henricotiae (Che), is more aggressive, heat-tolerant, and less sensitive to fungicides than its globally widespread counterpart, C. pseudonaviculata (Cps). However, the genetic underpinnings for these phenotypic differences are not well understood. We conducted a comparative genomic analysis of 10 Che and 69 Cps isolates and generated the first in planta transcriptomic dataset for these pathogens. Analysis of inoculated tissue revealed 5,073 differentially expressed genes (DEGs). While many DEGs were shared (3,124), the representative Che isolate expressed a larger (1,038 vs. 908 significantly upregulated in planta [UIP] genes) and more diverse (896 vs. 70 characterized genes) unique UIP genes than the representative Cps isolate, with most unique virulence-associated genes upregulated at 10 days post inoculation. This expanded arsenal of virulence genes in Che included numerous unique CAZymes, heat shock proteins, and host immune evasion genes, providing a genetic basis for its heightened virulence and thermal tolerance. Among predicted secondary metabolite biosynthetic genes, five genes were shared and two were UIP only in Che. Among the most similar known clusters, putative clavaric acid and choline biosynthesis genes were UIP in both species. Furthermore, four Cps genomic groups indicated potential population-level divergence. This work not only elucidates the potential genetic basis for Che's aggressiveness but also provides candidate genes for future infiltration assays to help identify resistant boxwood germplasm and for developing improved Che-specific rapid diagnostic assays.