bEndohyphal 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. Finally, we manipulate conditions to optimize reintroduction in a focal EHB-fungal association. We show that EHB infections were initiated and maintained more often under low-nutrient culture conditions and when EHB and fungal hyphae were washed with MgCl 2 prior to reassociation. Our study provides new methods for experimental assessment of the effects of EHB on fungal phenotypes and shows how the identity of the fungal host and growth conditions can define the establishment of these widespread and important symbioses.A s appreciation for their diversity and importance grows, plant microbiomes are increasingly of interest for diverse medical, industrial, and agricultural applications (1, 2). However, many plant-associated microbes remain undescribed (3, 4) and/or are found in association with, or as symbionts of, other microorganisms (5-14, 49-52). One example of these microbial symbioses is that between plant-associated fungi and their bacterial endosymbionts (endobacteria, endofungal bacteria, or endohyphal bacteria [EHB]). Recent studies have indicated that EHB are widespread in rhizosphere fungi from diverse fungal phyla (e.g., mycorrhizal and pathogenic fungi from the Basidiomycota, Glomeromycota, and Mucoromycotina [6][7][8][9][10][11][12][13]) and in the highly diverse Ascomycota that infect roots, stems, and leaves as endophytes (14) (class 3 endophytes, sensu [15]). However, functional relationships have been studied for only a few associations, limiting inferences regarding the scope and potential importance of EHB-fungal associations in ecological interactions and human applications (but see references 16 and 17).Most studies aimed at understanding functional relationships between EHB and fungi have focused on root-associated fungi, especially arbuscular mycorrhizal fungi in the Glomeromycota and plant-pathogenic Rhizopus in the Mucoromycotina (6-11, 18-23). EHB in rhizosphere fungi are often vertically transmitted and host specific, and they frequently maintain obligate relationships with their hosts (18)(19)(20). Although EHB can alter the phenotypes of root-associated fungi and profoundly i...

The ecological modes of fungi are shaped not only by their intrinsic features and the environment in which they occur, but also by their interactions with diverse microbes. Here we explore the ecological and genomic features of diverse bacterial endosymbionts-endohyphal bacteria-that together are emerging as major determinants of fungal phenotypes and plant-fungi interactions. We first provide a historical perspective on the study of endohyphal bacteria. We then propose a functional classification of three main groups, providing an overview of their genomic, phylogenetic, and ecological traits. Last, we explore frontiers in the study of endohyphal bacteria, with special attention to those facultative and horizontally transmitted bacteria that associate with some of the most diverse lineages of fungi. Overall, our aim is to synthesize the rich literature from nearly 50 years of studies on endohyphal bacteria as a means to highlight potential applications and new research directions.

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