Cytospora canker (CC) is among the most important diseases in conifer trees (Picea spp., mainly). This disease poses a significant risk factor for forest health, potentially leading to economic losses for wood producers. To provide a genomic basis of the CC pathogenesis, the genomes of two Cytospora species associated with the disease were sequenced and further analyzed within a set of Diaporthales species. The first species was identified as C. piceae. The second was described as C. piceicola sp. nov. based on morphological characteristics and multi-gene phylogenetic analysis. The novel species is sister to other Cytospora species isolated from conifers. Here, we report 39.7 and 43.8 Mb highly contiguous genome assemblies of C. piceae EI-19(A) and C. piceicola EI-20, respectively, obtained using Illumina sequencing technology. Despite notably different genome sizes, these species share the main genome characteristics, such as predicted gene number (10,862 and 10,742) and assembly completeness (97.6% and 98.1%). A wide range of genes encoding carbohydrate-active enzymes, secondary metabolite biosynthesis clusters, and secreted effectors were found. Multiple experimentally validated virulence genes were also identified in the studied species. The defined arsenals of enzymes and effectors generally relate to the hemibiotrophic lifestyle with a capability to switch to biotrophy. The obtained evidence also supports that C. piceae EI-19(A) and C. piceicola EI-20 can cause severe canker disease symptoms in Picea spp. specifically. It was additionally observed that the strains of C. piceae may have different pathogenicity and virulence characteristics based on the analyses of predicted secondary metabolite complements, effectomes, and virulence-related genes. Phylogenomic analysis and timetree estimations indicated that divergence of the studied species may have occurred relatively late, 11-10 million years ago. Compared to other members of Diaporthales, C. piceae EI-19(A) and C. piceicola EI-20 implied a moderate rate of gene contraction, but the latter experienced significant gene loss that can additionally support host specificity attributed to these species. But uncovered gene contraction events may point out potential lifestyle differentiation and host shift of the studied species. It was revealed that EI-19(A) and C. piceicola EI-20 carry distinct secretomes and effectomes among Diaporthales species. This feature can indicate a species lifestyle and pathogenicity potential. These findings highlight potential targets for identification and/or detection of pathogenic Cytospora in conifers. The introduced draft genome sequences of C. piceae and C. piceicola can be employed as tools to understand basic genetics and pathogenicity mechanisms of fungal species causing canker disease in woody plants. The identified pathogenicity and virulence-related genes would serve as potential candidates for host-induced gene silencing aimed at making plant hosts more resistant to pathogenic species. Furthermore, the comparative genomics component of the study will facilitate the functional analysis of the genes of unknown function in all fungal pathogens.
