Given a shared toolbox of pathogenicity-related genes among a set of species, why is one species a biotroph and specialist while another is a necrotroph and generalist? Is it the result of selection on primary sequence, or on proteins, or alternatively, differences in the timing and magnitude of gene expression? The Sclerotiniaceae (Ascomycota, Leotiomycetes, Helotiales) is a relatively recently evolved family of fungi whose members include host generalists and host specialists, and the spectrum of trophic types. Based on a phylogeny inferred from three, presumably evolutionarily conserved housekeeping genes, the common ancestor of the Sclerotiniaceae was necrotrophic, with at least two shifts from necrotrophy to biotrophy. Phylogenies inferred from eight pathogenicity-related genes, involved in cell wall degradation and the oxalic acid pathway, were incongruent with the presumably neutral phylogeny. Site-specific likelihood analyses, which estimate the rate of nonsynonymous to synonymous substitutions (dN/dS), showed evidence for purifying selection acting on all pathogenicity-related genes, and positive selection on sites within five of eight genes. Rate-specific likelihood analyses showed no differences in dN/dS rates between necrotrophs and biotrophs, and between host generalists and host specialists, indicating that selection acting on the genes does not drive divergence toward changes in trophic type or host association. In vitro screens for oxalic acid production demonstrated that all necrotrophic generalists produce oxalic acid by 72 hours, while production was either absent or delayed among biotrophs and host specialists. This pattern was also observed during the course of Arabidopsis thaliana infection, in which large spikes of expression were seen in the oxalic acid pathway-related gene, oah, within eight hours of inoculation among necrotrophic generalists only. Results suggest that necrotrophic generalists can be distinguished from biotrophs and host specialists in the Sclerotiniaceae by the ability to produce abundant amounts of oxalic acid early in infection and to cause large proliferating lesions on A. thaliana.

Given a shared toolbox of pathogenicity-related genes among a set of species, why is one species a biotroph and specialist while another is a necrotroph and generalist? Is it the result of selection on primary sequence, or on proteins, or alternatively, differences in the timing and magnitude of gene expression? The Sclerotiniaceae (Ascomycota, Leotiomycetes, Helotiales) is a relatively recently evolved family of fungi whose members include host generalists and host specialists, and the spectrum of trophic types. Based on a phylogeny inferred from three, presumably evolutionarily conserved housekeeping genes, the common ancestor of the Sclerotiniaceae was necrotrophic, with at least two shifts from necrotrophy to biotrophy. Phylogenies inferred from eight pathogenicity-related genes, involved in cell wall degradation and the oxalic acid pathway, were incongruent with the presumably neutral phylogeny. Site-specific likelihood analyses, which estimate the rate of nonsynonymous to synonymous substitutions (dN/dS), showed evidence for purifying selection acting on all pathogenicity-related genes, and positive selection on sites within five of eight genes. Rate-specific likelihood analyses showed no differences in dN/dS rates between necrotrophs and biotrophs, and between host generalists and host specialists, indicating that selection acting on the genes does not drive divergence toward changes in trophic type or host association. In vitro screens for oxalic acid production demonstrated that all necrotrophic generalists produce oxalic acid by 72 hours, while production was either absent or delayed among biotrophs and host specialists. This pattern was also observed during the course of Arabidopsis thaliana infection, in which large spikes of expression were seen in the oxalic acid pathway-related gene, oah, within eight hours of inoculation among necrotrophic generalists only. Results suggest that necrotrophic generalists can be distinguished from biotrophs and host specialists in the Sclerotiniaceae by the ability to produce abundant amounts of oxalic acid early in infection and to cause large proliferating lesions on A. thaliana.