Detection of a novel intracellular microbiome hosted in arbuscular mycorrhizal fungi

Abstract

Arbuscular mycorrhizal fungi (AMF) are important members of the plant microbiome. They are obligate biotrophs that colonize the roots of most land plants and enhance host nutrient acquisition. Many AMF themselves harbor endobacteria in their hyphae and spores. Two types of endobacteria are known in Glomeromycota: rod-shaped Gram-negative Candidatus Glomeribacter gigasporarum, CaGg, limited in distribution to members of the Gigasporaceae family, and coccoid Mollicutes-related endobacteria, Mre, widely distributed across different lineages of AMF. The goal of the present study is to investigate the patterns of distribution and coexistence of the two endosymbionts, CaGg and Mre, in spore samples of several strains of Gigaspora margarita. Based on previous observations, we hypothesized that some AMF could host populations of both endobacteria. To test this hypothesis, we performed an extensive investigation of both endosymbionts in G. margarita spores sampled from Cameroonian soils as well as in the Japanese G. margarita MAFF520054 isolate using different approaches (molecular phylotyping, electron microscopy, fluorescence in situ hybridization and quantitative real-time PCR). We found that a single AMF host can harbour both types of endobacteria, with Mre population being more abundant, variable and prone to recombination than the CaGg one. Both endosymbionts seem to retain their genetic and lifestyle peculiarities regardless of whether they colonize the host alone or together. These findings show for the first time that fungi support an intracellular bacterial microbiome, in which distinct types of endobacteria coexist in a single cell.

Symbiosis with an endobacterium increases the fitness of a mycorrhizal fungus, raising its bioenergetic potential

“…Confocal and electron microscopy suggest that bacteria are diluted along the intraradical symbiotic mycelium (Bianciotto et al, 1996), while spores (Figure 1) act as a reservoir of these microbial communities (Desirò et al, 2014). This result opens the question of what advantages the endobacterium provides to its fungal host during this phase.…”

“…The spores were then crushed with a coverslip, incubated in the dark for at least 5 min, and observed under a Leica (Wetzlar, Germany) TCSSP2 confocal microscope (excitation 488 nm; emission 520 nm) to detect the endobacteria. To perform ultrastructural observations, parallel sets of single spores were processed by high-pressure freezing followed by freeze substitution, as described in Desirò et al (2014). After cutting and counterstaining, thin sections were observed under a transmission electron microscope Philips CM10 (Amsterdam, The Netherlands).…”

Diversity, Specificity, and Phylogenetic Relationships of Endohyphal Bacteria in Fungi That Inhabit Tropical Seeds and Leaves

“…Overall, we generated 22 alignments that together contained 284 sequences representing EHB from our surveys. Our data sets also included 61 EHB sequences identified previously from endophytic Ascomycota (Hoffman and Arnold, 2010), 28 EHB sequences from root-associated Mucoromycotina, Mortierellomycotina, and Glomeromycota (Bianciotto et al, 2003;Partida-Martinez and Hertweck, 2005;Partida-Martinez et al, 2007a;Naumann et al, 2010;Sato et al, 2010;Lackner et al, 2011;Desirò et al, 2014Desirò et al, , 2015, and 470 non-EHB reference sequences from GenBank representing the type or otherwise named strains with greatest homology to our unclassified EHB (Supplementary Table 4). …”

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

“…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).…”