Comparative pangenomic insights into the distinct evolution of virulence factors among grapevine trunk pathogens

The permanent organs of grapevines ( V. vinifera L.), like other woody perennials, are colonized by various unrelated pathogenic ascomycete fungi secreting cell wall-degrading enzymes and phytotoxic secondary metabolites that contribute to host damage and disease symptoms. Trunk pathogens differ in the symptoms they induce and the extent and speed of damage. Isolates of the same species often display a wide virulence range, even within the same vineyard. This study focuses on Eutypa lata , Neofusicoccum parvum , and Phaeoacremonium minimum , causal agents of Eutypa dieback, Botryosphaeria dieback, and Esca, respectively. We sequenced fifty isolates from viticulture regions worldwide and built nucleotide-level, reference-free pangenomes for each species. Through examining genomic diversity and pangenome structure, we analyzed intraspecific conservation and variability of putative virulence factors, focusing on functions under positive selection, and recent gene-family dynamics of contraction and expansion. Our findings reveal contrasting distributions of putative virulence factors in the core, dispensable, and private genomes of each pangenome. For example, CAZymes were prevalent in the core genomes of each pangenome, whereas biosynthetic gene clusters were prevalent in the dispensable genomes of E. lata and P. minimum . The dispensable fractions were also enriched in Gypsy transposable elements and virulence factors under positive selection (polyketide synthases genes in E. lata and P. minimum glycosyltransferases in N. parvum ). Our findings underscore the complexity of the genomic architecture in each species and provide insights into their adaptive strategies, enhancing our understanding of the underlying mechanisms of virulence.