Absence of genome reduction in diverse, facultative endohyphal bacteria.

Fungi interact closely with bacteria, both on the surfaces of the hyphae and within their living tissues (i.e. endohyphal bacteria, EHB). These EHB can be obligate or facultative symbionts and can mediate diverse phenotypic traits in their hosts. Although EHB have been observed in many lineages of fungi, it remains unclear how widespread and general these associations are, and whether there are unifying ecological and genomic features can be found across EHB strains as a whole. We cultured 11 bacterial strains after they emerged from the hyphae of diverse Ascomycota that were isolated as foliar endophytes of cupressaceous trees, and generated nearly complete genome sequences for all. Unlike the genomes of largely obligate EHB, the genomes of these facultative EHB resembled those of closely related strains isolated from environmental sources. Although all analysed genomes encoded structures that could be used to interact with eukaryotic hosts, pathways previously implicated in maintenance and establishment of EHB symbiosis were not universally present across all strains. Independent isolation of two nearly identical pairs of strains from different classes of fungi, coupled with recent experimental evidence, suggests horizontal transfer of EHB across endophytic hosts. Given the potential for EHB to influence fungal phenotypes, these genomes could shed light on the mechanisms of plant growth promotion or stress mitigation by fungal endophytes during the symbiotic phase, as well as degradation of plant material during the saprotrophic phase. As such, these findings contribute to the illumination of a new dimension of functional biodiversity in fungi.

Isolation of Endohyphal Bacteria from Foliar Ascomycota and In Vitro Establishment of Their Symbiotic Associations.

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. 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 MgCl2 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.

Fungal endophytes in aboveground tissues of desert plants: infrequent in culture, but highly diverse and distinctive symbionts.

In hot deserts, plants cope with aridity, high temperatures, and nutrient-poor soils with morphological and biochemical adaptations that encompass intimate microbial symbioses. Whereas the root microbiomes of arid-land plants have received increasing attention, factors influencing assemblages of symbionts in aboveground tissues have not been evaluated for many woody plants that flourish in desert environments. We evaluated the diversity, host affiliations, and distributions of endophytic fungi associated with photosynthetic tissues of desert trees and shrubs, focusing on nonsucculent woody plants in the species-rich Sonoran Desert. To inform our strength of inference, we evaluated the effects of two different nutrient media, incubation temperatures, and collection seasons on the apparent structure of endophyte assemblages. Analysis of >22,000 tissue segments revealed that endophytes were isolated four times more frequently from photosynthetic stems than leaves. Isolation frequency was lower than expected given the latitude of the study region and varied among species a function of sampling site and abiotic factors. However, endophytes were very species-rich and phylogenetically diverse, consistent with less arid sites of a similar latitudinal position. Community composition differed among host species, but not as a function of tissue type, sampling site, sampling month, or exposure. Estimates of abundance, diversity, and composition were not influenced by isolation medium or incubation temperature. Phylogenetic analyses of the most commonly isolated genus (Preussia) revealed multiple evolutionary origins of desert-plant endophytism and little phylogenetic structure with regard to seasonality, tissue preference, or optimal temperatures and nutrients for growth in vitro. Together, these results provide insight into endophytic symbioses in desert-plant communities and can be used to optimize strategies for capturing endophyte biodiversity at regional scales.