Alessandro Desirò

Detection of a novel intracellular microbiome hosted in arbuscular mycorrhizal fungi

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.

Fungal symbioses in hornworts: a chequered history

Hornworts are considered the sister group to vascular plants, but their fungal associations remain largely unexplored. The ancestral symbiotic condition for all plants is, nonetheless, widely assumed to be arbuscular mycorrhizal with Glomeromycota fungi. Owing to a recent report of other fungi in some non-vascular plants, here we investigate the fungi associated with diverse hornworts worldwide, using electron microscopy and molecular phylogenetics. We found that both Glomeromycota and Mucoromycotina fungi can form symbioses with most hornworts, often simultaneously. This discovery indicates that ancient terrestrial plants relied on a wider and more versatile symbiotic repertoire than previously thought, and it highlights the so far unappreciated ecological and evolutionary role of Mucoromycotina fungi.

Endogone, one of the oldest plant‐associated fungi, host unique Mollicutes‐related endobacteria

Glomeromycota have been considered the most ancient group of fungi capable of positively interacting with plants for many years. Recently, other basal fungi, the Endogone Mucoromycotina fungi, have been identified as novel plant symbionts, challenging the paradigm of Glomeromycota as the unique ancestral symbionts of land plants. Glomeromycota are known to host endobacteria and recent evidences show that also some Mucoromycotina contain endobacteria. In order to examine similarities between basal groups of plant-associated fungi, we tested whether Endogone contained endobacteria. Twenty-nine Endogone were investigated in order to identify Mollicutes-related endobacteria (Mre). Fruiting bodies were processed for transmission electron microscopy and molecularly investigated using fungal and Mre-specific primers. We demonstrate that Mre are present inside 13 out of 29 Endogone: endobacteria are directly embedded in the fungal cytoplasm and their 16S rDNA sequences cluster together with the ones retrieved from Glomeromycota, forming, however, a separate new clade. Our findings provide new insights on the evolutionary relations between Glomeromycota, Mucoromycotina and endobacteria, raising new questions on the role of these still enigmatic microbes in the ecology, evolution and diversification of their fungal hosts during the history of plant-fungal symbiosis.

Comparative genomics of Mortierella elongata and its bacterial endosymbiont Mycoavidus cysteinexigens

Endosymbiosis of bacteria by eukaryotes is a defining feature of cellular evolution. In addition to well-known bacterial origins for mitochondria and chloroplasts, multiple origins of bacterial endosymbiosis are known within the cells of diverse animals, plants and fungi. Early-diverging lineages of terrestrial fungi harbor endosymbiotic bacteria belonging to the Burkholderiaceae. We sequenced the metagenome of the soil-inhabiting fungus Mortierella elongata and assembled the complete circular chromosome of its endosymbiont, Mycoavidus cysteinexigens, which we place within a lineage of endofungal symbionts that are sister clade to Burkholderia. The genome of M. elongata strain AG77 features a core set of primary metabolic pathways for degradation of simple carbohydrates and lipid biosynthesis, while the M. cysteinexigens (AG77) genome is reduced in size and function. Experiments using antibiotics to cure the endobacterium from the host demonstrate that the fungal host metabolism is highly modulated by presence/absence of M. cysteinexigens. Independent comparative phylogenomic analyses of fungal and bacterial genomes are consistent with an ancient origin for M. elongata - M. cysteinexigens symbiosis, most likely over 350 million years ago and concomitant with the terrestrialization of Earth and diversification of land fungi and plants.

Who lives in a fungus? The diversity, origins and functions of fungal endobacteria living in Mucoromycota

Bacterial interactions with plants and animals have been examined for many years; differently, only with the new millennium the study of bacterial–fungal interactions blossomed, becoming a new field of microbiology with relevance to microbial ecology, human health and biotechnology. Bacteria and fungi interact at different levels and bacterial endosymbionts, which dwell inside fungal cells, provide the most intimate example. Bacterial endosymbionts mostly occur in fungi of the phylum Mucoromycota and include Betaproteobacteria (Burkhoderia-related) and Mollicutes (Mycoplasma-related). Based on phylogenomics and estimations of divergence time, we hypothesized two different scenarios for the origin of these interactions (early vs late bacterial invasion). Sequencing of the genomes of fungal endobacteria revealed a significant reduction in genome size, particularly in endosymbionts of Glomeromycotina, as expected by their uncultivability and host dependency. Similar to endobacteria of insects, the endobacteria of fungi show a range of behaviours from mutualism to antagonism. Emerging results suggest that some benefits given by the endobacteria to their plant-associated fungal host may propagate to the interacting plant, giving rise to a three-level inter-domain interaction.

Mollicutes‐related endobacteria thrive inside liverwort‐associated arbuscular mycorrhizal fungi

Arbuscular mycorrhizal fungi (AMF) can host Gram-positive endobacteria (BLOs) in their cytoplasm. These have been identified as Mollicutes-related microbes based on an inventory of AMF spores from fungal collections. Bacteria-like organisms (BLOs) of unknown identity have also been reported in the cytoplasm of AMF associated with liverworts, the earliest-diverged extant lineage of land plants. A combination of morphological, molecular and phylogenetic analyses revealed that three samples of two liverwort species (Conocephalum conicum and Lunularia cruciata) growing spontaneously in a botanical garden harboured AMF belonging to Glomerales, and these, in turn, hosted coccoid BLOs. 16S rDNA sequences from these BLOs clustered with the Mollicutes sequences identified from the spore collections but revealed the presence of novel phylotypes. Electron microscopy and fluorescence in situ hybridization (FISH) confirmed the presence of BLOs inside the cytoplasm of AMF hyphae colonizing the liverwort thalli. The high genetic variability of BLOs in liverwort-AMF associations thriving in the same ecological niche raises questions about the mechanisms underlying such diversity.

Multigene phylogeny of Endogonales, an early diverging lineage of fungi associated with plants

Endogonales is a lineage of early diverging fungi within Mucoromycota. Many species in this order produce small sporophores (“sporocarps”) containing a large number of zygospores, and many species form symbioses with plants. However, due to limited collections, subtle morphological differentiation, difficulties in growing these organisms in vitro, and idiosyncrasies in their rDNA that make PCR amplification difficult, the systematics and character evolution of these fungi have been challenging to resolve. To overcome these challenges we generated a multigene phylogeny of Endogonales using sporophores collected over the past three decades from four continents. Our results show that Endogonales harbour significant undescribed diversity and form two deeply divergent and well-supported phylogenetic clades, which we delimit as the families Endogonaceae and Densosporaceae fam. nov. The family Densosporaceae consists of the genus Densospora, Sphaerocreas pubescens, and many diverse lineages known only from environmental DNA sequences of plant-endosymbiotic fungi. Within Endogonaceae there are two clades. One corresponds to Endogone and includes the type species, E. pisiformis. Species of Endogone are characterized by above- and below-ground sporophores, a hollow and infolded sporophore form, a loose zygosporangial hyphal mantle, homogeneous gametangia, and an enigmatic trophic mode with no evidence of ectomycorrhizal association for most species. For the other clade we introduce a new generic name, Jimgerdemannia gen. nov. Members of that genus (J. flammicorona and J. lactiflua species complexes, and an undescribed species) are characterized by hypogeous sporophores with a solid gleba, a well-developed zygosporangial hyphal mantle, heterogeneous gametangia, and an ectomycorrhizal trophic mode. Future studies on Densosporaceae and Endogonaceae will be important for understanding fungal innovations including evolution of macroscopic sporophores and symbioses with plants.

ITS fungal barcoding primers vs 18S AMF-specific primers reveal similar AMF-based diversity patterns in roots and soils of three mountain vineyards

ITS primers commonly used to describe soil fungi are flawed for AMF although it is unknown the extent to which they distort the interpretation of community patterns. Here, we focus on how the use of a specific ITS2 fungal barcoding primer pair biased for AMF changes the interpretation of AMF community patterns from three mountain vineyards compared to a novel AMF-specific approach on the 18S. We found that although discrepancies were present in the taxonomic composition of the two resulting datasets, the estimation of diversity patterns among AMF communities was similar and resulted in both primer systems being able to correctly assess the community-structuring effect of location, compartment (root vs. soil) and environment. Both methodologies made it possible to detect the same alpha-diversity trend among the locations under study but not between root and soil transects. We show that the ITS2 primer system for fungal barcoding provides a good estimate of both AMF community structure and relation to environmental variables. However, this primer system does not fit in with cross-compartment surveys (roots vs. soil) as it can underestimate AMF diversity in soil samples. When specifically focusing on AMF, the 18S primer system resulted in wide coverage and marginal non-target amplification.

Mycoplasma-related endobacteria within Mortierellomycotina fungi: diversity, distribution and functional insights into their lifestyle

Bacterial interactions with animals and plants have been examined for over a century; by contrast, the study of bacterial–fungal interactions has received less attention. Bacteria interact with fungi in diverse ways, and endobacteria that reside inside fungal cells represent the most intimate interaction. The most significant bacterial endosymbionts that have been studied are associated with Mucoromycota and include two main groups: Burkholderia-related and Mycoplasma-related endobacteria (MRE). Examples of Burkholderia-related endobacteria have been reported in the three Mucoromycota subphyla. By contrast, MRE have only been identified in Glomeromycotina and Mucoromycotina. This study aims to understand whether MRE dwell in Mortierellomycotina and, if so, to determine their impact on the fungal host. We carried out a large-scale screening of 394 Mortierellomycotina strains and employed a combination of microscopy, molecular phylogeny, next-generation sequencing and qPCR. We detected MRE in 12 strains. These endosymbionts represent novel bacterial phylotypes and show evidence of recombination. Their presence in Mortierellomycotina demonstrates that MRE occur within fungi across Mucoromycota and they may have lived in their common ancestor. We cured the fungus of its endosymbionts with antibiotics and observed improved biomass production in isogenic lines lacking MRE, demonstrating that these endobacteria impose some fitness costs to their fungal host. Here we provided the first functional insights into the lifestyle of MRE. Our findings indicate that MRE may be antagonistic to their fungal hosts, and adapted to a non-lethal parasitic lifestyle in the mycelium of Mucoromycota. However, context-dependent adaptive benefits to their host at minimal cost cannot not be excluded. Finally, we conclude that Mortierellomycotina represent attractive model organisms for exploring interactions between MRE and fungi.

Investigating the Endobacteria Which Thrive in Arbuscular Mycorrhizal Fungi

The study of the so-called unculturable bacteria is still considered a challenging task. However, given recent improvements in the sensitivity of culture-free approaches, the identification and characterization of such microbes in complex biological samples is now possible. In this chapter we report how endobacteria thriving inside arbuscular mycorrhizal fungi (AMF), which are themselves obligate biotrophs of plants, can be studied using a combination of in vitro culture, molecular biology, and microscopy techniques.