Host–pathogen infections and possible effects on co-evolutionary patterns depend on the genotypes of both host and pathogen. Obligate fungal pathogens of plants are often characterized by host–pathogen genotype-by-genotype (GxG) interactions, but whether these patterns exist in obligate insect fungal pathogens is unclear. We take advantage of the obligate insect pathogenic fungus Entomophthora muscae, where individual isolates are specific to different dipteran host species in nature but can cross-infect multiple fly species in the laboratory. We collected three new isolates of E. muscae from Drosophila species. Phylogenetic analysis showed that Drosophila-isolated E. muscae represents a distinct geographically widespread Drosophila lineage compared to the house fly (Musca domestica) or Delia species-isolated E. muscae. We used the three new E. muscae isolates from Drosophila spp. together with a genetically distinct E. muscae isolate from house flies and assessed their virulence in a cross-infection experiment using one house fly, three Drosophila suzukii, and two D. melanogaster genotypes as hosts. All fungal isolates successfully infected hosts, induced behavioural manipulation, sporulated in all fly hosts, and differed in virulence between host genotypes, revealing GxG interactions. While house flies were most susceptible to fungal infection with 99% mortality, we found a lower virulence of 49% and 25% mortality in D. melanogaster and D. suzukii genotypes, respectively. Furthermore, all isolates harboured a specific mycovirus (family Iflaviridae), but co-phylogenetic branching patterns did not support fungus–virus co-speciation. We show that the genetic makeup of both fungal pathogen and fly host influence E. muscae infectivity, confirming GxG interactions in obligate fly fungal pathogens.
