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Endohyphal bacteria (EHB) can influence fungal phenotypes and shape the outcomes of plant-fungal interactions. Previous work has suggested that EHB form facultative associations with many foliar fungi in the Ascomycota. These bacteria can be isolated in culture, and fungi can be cured of EHB using antibiotics. Here, we present methods for successfully introducing EHB into axenic mycelia of strains representing two classes of Ascomycota. We first establish in vitro conditions favoring reintroduction of two strains of EHB (Luteibacter sp.) into axenic cultures of their original fungal hosts, focusing on fungi isolated from healthy plant tissue as endophytes: Microdiplodia sp. (Dothideomycetes) and Pestalotiopsis sp. (Sordariomycetes). We then demonstrate that these EHB can be introduced into a novel fungal host under the same conditions, successfully transferring EHB between fungi representing different classes …