Ecology and evolution of algal-fungal symbioses.

Ecological interactions and symbiosis between algae and fungi are ancient, widespread, and diverse with many independent origins. The heterotrophic constraint on fungal nutrition drives fungal interactions with autotrophic organisms, including algae. While ancestors of modern fungi may have evolved as parasites of algae, there remains a latent ability in algae to detect and respond to fungi through a range of symbioses that are witnessed today in the astounding diversity of lichens, associations with corticoid and polypore fungi, and endophytic associations with macroalgae. Research into algal-fungal interactions and biotechnological innovation have the potential to improve our understanding of their diversity and functions in natural systems, and to harness this knowledge to develop sustainable and novel approaches for producing food, energy, and bioproducts.

Multilevel analysis between Physcomitrium patens and Mortierellaceae endophytes explores potential long-standing interaction among land plants and fungi.

The model moss species Physcomitrium patens has long been used for studying divergence of land plants spanning from bryophytes to angiosperms. In addition to its phylogenetic relationships, the limited number of differential tissues, and comparable morphology to the earliest embryophytes provide a system to represent basic plant architecture. Based on plant-fungal interactions today, it is hypothesized these kingdoms have a long-standing relationship, predating plant terrestrialization. Mortierellaceae have origins diverging from other land fungi paralleling bryophyte divergence, are related to arbuscular mycorrhizal fungi but are free-living, observed to interact with plants, and can be found in moss microbiomes globally. Due to their parallel origins, we assess here how two Mortierellaceae species, Linnemannia elongata and Benniella erionia, interact with P. patens in coculture. We also assess how Mollicute-related or Burkholderia-related endobacterial symbionts (MRE or BRE) of these fungi impact plant response. Coculture interactions are investigated through high-throughput phenomics, microscopy, RNA-sequencing, differential expression profiling, gene ontology enrichment, and comparisons among 99 other P. patens transcriptomic studies. Here we present new high-throughput approaches for measuring P. patens growth, identify novel expression of over 800 genes that are not expressed on traditional agar media, identify subtle interactions between P. patens and Mortierellaceae, and observe changes to plant-fungal interactions dependent on whether MRE or BRE are present. Our study provides insights into how plants and fungal partners may have interacted based on their communications observed today as well as identifying L. elongata and B. erionia as modern fungal endophytes with P. patens.

Associated bacterial communities, confrontation studies, and comparative genomics reveal important interactions between Morchella with Pseudomonas spp.

Members of the fungal genus Morchella are widely known for their important ecological roles and significant economic value. In this study, we used amplicon and genome sequencing to characterize bacterial communities associated with sexual fruiting bodies from wild specimens, as well as vegetative mycelium and sclerotia obtained from Morchella isolates grown in vitro. These investigations included diverse representatives from both Elata and Esculenta Morchella clades. Unique bacterial community compositions were observed across the various structures examined, both within and across individual Morchella isolates or specimens. However, specific bacterial taxa were frequently detected in association with certain structures, providing support for an associated core bacterial community. Bacteria from the genus Pseudomonas and Ralstonia constituted the core bacterial associates of Morchella mycelia and sclerotia, while other genera (e.g., Pedobacter spp., Deviosa spp., and Bradyrhizobium spp.) constituted the core bacterial community of fruiting bodies. Furthermore, the importance of Pseudomonas as a key member of the bacteriome was supported by the isolation of several Pseudomonas strains from mycelia during in vitro cultivation. Four of the six mycelial-derived Pseudomonas isolates shared 16S rDNA sequence identity with amplicon sequences recovered directly from the examined fungal structures. Distinct interaction phenotypes (antagonistic or neutral) were observed in confrontation assays between these bacteria and various Morchella isolates. Genome sequences obtained from these Pseudomonas isolates revealed intriguing differences in gene content and annotated functions, specifically with respect to toxin-antitoxin systems, cell adhesion, chitinases, and insecticidal toxins. These genetic differences correlated with the interaction phenotypes. This study provides evidence that Pseudomonas spp. are frequently associated with Morchella and these associations may greatly impact fungal physiology.

Characterizing microbial communities associated with northern root-knot nematode (Meloidogyne hapla) occurrence and soil health.

The northern root-knot nematode (Meloidogyne hapla) causes extensive damage to agricultural crops globally. In addition, M. hapla populations with no known genetic or morphological differences exhibit parasitic variability (PV) or reproductive potential based on soil type. However, why M. hapla populations from mineral soil with degraded soil health conditions have a higher PV than populations from muck soil is unknown. To improve our understanding of soil bio-physicochemical conditions in the environment where M. hapla populations exhibited PV, this study characterized the soil microbial community and core- and indicator-species structure associated with M. hapla occurrence and soil health conditions in 15 Michigan mineral and muck vegetable production fields. Bacterial and fungal communities in soils from where nematodes were isolated were characterized with high throughput sequencing of 16S and internal transcribed spacer (ITS) rDNA. Our results showed that M. hapla-infested, as well as disturbed and degraded muck fields, had lower bacterial diversity (observed richness and Shannon) compared to corresponding mineral soil fields or non-infested mineral fields. Bacterial and fungal community abundance varied by soil group, soil health conditions, and/or M. hapla occurrence. A core microbial community was found to consist of 39 bacterial and 44 fungal sub-operational taxonomic units (OTUs) across all fields. In addition, 25 bacteria were resolved as indicator OTUs associated with M. hapla presence or absence, and 1,065 bacteria as indicator OTUs associated with soil health conditions. Out of the 1,065 bacterial OTUs, 73.9% indicated stable soil health, 8.4% disturbed, and 0.4% degraded condition; no indicators were common to the three categories. Collectively, these results provide a foundation for an in-depth understanding of the environment where M. hapla exists and conditions associated with parasitic variability.

Connecting the multiple dimensions of global soil fungal diversity.

How the multiple facets of soil fungal diversity vary worldwide remains virtually unknown, hindering the management of this essential species-rich group. By sequencing high-resolution DNA markers in over 4000 topsoil samples from natural and human-altered ecosystems across all continents, we illustrate the distributions and drivers of different levels of taxonomic and phylogenetic diversity of fungi and their ecological groups. We show the impact of precipitation and temperature interactions on local fungal species richness (alpha diversity) across different climates. Our findings reveal how temperature drives fungal compositional turnover (beta diversity) and phylogenetic diversity, linking them with regional species richness (gamma diversity). We integrate fungi into the principles of global biodiversity distribution and present detailed maps for biodiversity conservation and modeling of global ecological processes.

Contrasting effects of bioenergy crops on biodiversity.

Agriculture is driving biodiversity loss, and future bioenergy cropping systems have the potential to ameliorate or exacerbate these effects. Using a long-term experimental array of 10 bioenergy cropping systems, we quantified diversity of plants, invertebrates, vertebrates, and microbes in each crop. For many taxonomic groups, alternative annual cropping systems provided no biodiversity benefits when compared to corn (the business-as-usual bioenergy crop in the United States), and simple perennial grass-based systems provided only modest gains. In contrast, for most animal groups, richness in plant-diverse perennial systems was much higher than in annual crops or simple perennial systems. Microbial richness patterns were more eclectic, although some groups responded positively to plant diversity. Future agricultural landscapes incorporating plant-diverse perennial bioenergy cropping systems could be of high conservation value. However, increased use of annual crops will continue to have negative effects, and simple perennial grass systems may provide little improvement over annual crops.

Terpenes modulate bacterial and fungal growth and sorghum rhizobiome communities.

Terpenes are among the oldest and largest class of plant-specialized bioproducts that are known to affect plant development, adaptation, and biological interactions. While their biosynthesis, evolution, and function in aboveground interactions with insects and individual microbial species are well studied, how different terpenes impact plant microbiomes belowground is much less understood. Here we designed an experiment to assess how belowground exogenous applications of monoterpenes (1,8-cineole and linalool) and a sesquiterpene (nerolidol) delivered through an artificial root system impacted its belowground bacterial and fungal microbiome. We found that the terpene applications had significant and variable impacts on bacterial and fungal communities, depending on terpene class and concentration; however, these impacts were localized to the artificial root system and the fungal rhizosphere. We complemented this experiment with pure culture bioassays on responsive bacteria and fungi isolated from the sorghum rhizobiome. Overall, higher concentrations (200 µM) of nerolidol were inhibitory to Ferrovibrium and tested Firmicutes. While fungal isolates of Penicillium and Periconia were also more inhibited by higher concentrations (200 µM) of nerolidol, Clonostachys was enhanced at this higher level and together with Humicola was inhibited by the lower concentration tested (100 µM). On the other hand, 1,8-cineole had an inhibitory effect on Orbilia at both tested concentrations but had a promotive effect at 100 µM on Penicillium and Periconia. Similarly, linalool at 100 µM had significant growth promotion in Mortierella, but an inhibitory effect for Orbilia. Together, these results highlight the variable direct effects of terpenes on single microbial isolates and demonstrate the complexity of microbe-terpene interactions in the rhizobiome. IMPORTANCE Terpenes represent one of the largest and oldest classes of plant-specialized metabolism, but their role in the belowground microbiome is poorly understood. Here, we used a "rhizobox" mesocosm experimental set-up to supply different concentrations and classes of terpenes into the soil compartment with growing sorghum for 1 month to assess how these terpenes affect sorghum bacterial and fungal rhizobiome communities. Changes in bacterial and fungal communities between treatments belowground were characterized, followed by bioassays screening on bacterial and fungal isolates from the sorghum rhizosphere against terpenes to validate direct microbial responses. We found that microbial growth stimulatory and inhibitory effects were localized, terpene specific, dose dependent, and transient in time. This work paves the way for engineering terpene metabolisms in plant microbiomes for improved sustainable agriculture and bioenergy crop production.

Comparative genomics of Mollicutes-related endobacteria supports a late invasion into Mucoromycota fungi.

Diverse members of early-diverging Mucoromycota, including mycorrhizal taxa and soil-associated Mortierellaceae, are known to harbor Mollicutes-related endobacteria (MRE). It has been hypothesized that MRE were acquired by a common ancestor and transmitted vertically. Alternatively, MRE endosymbionts could have invaded after the divergence of Mucoromycota lineages and subsequently spread to new hosts horizontally. To better understand the evolutionary history of MRE symbionts, we generated and analyzed four complete MRE genomes from two Mortierellaceae genera: Linnemannia (MRE-L) and Benniella (MRE-B). These genomes include the smallest known of fungal endosymbionts and showed signals of a tight relationship with hosts including a reduced functional capacity and genes transferred from fungal hosts to MRE. Phylogenetic reconstruction including nine MRE from mycorrhizal fungi revealed that MRE-B genomes are more closely related to MRE from Glomeromycotina than MRE-L from the same host family. We posit that reductions in genome size, GC content, pseudogene content, and repeat content in MRE-L may reflect a longer-term relationship with their fungal hosts. These data indicate Linnemannia and Benniella MRE were likely acquired independently after their fungal hosts diverged from a common ancestor. This work expands upon foundational knowledge on minimal genomes and provides insights into the evolution of bacterial endosymbionts.

Convergent reductive evolution and host adaptation in Mycoavidus bacterial endosymbionts of Mortierellaceae fungi.

Intimate associations between fungi and intracellular bacterial endosymbionts are becoming increasingly well understood. Phylogenetic analyses demonstrate that bacterial endosymbionts of Mucoromycota fungi are related either to free-living Burkholderia or Mollicutes species. The so-called Burkholderia-related endosymbionts or BRE comprise Mycoavidus, Mycetohabitans and Candidatus Glomeribacter gigasporarum. These endosymbionts are marked by genome contraction thought to be associated with intracellular selection. However, the conclusions drawn thus far are based on a very small subset of endosymbiont genomes, and the mechanisms leading to genome streamlining are not well understood. The purpose of this study was to better understand how intracellular existence shapes Mycoavidus and BRE functionally at the genome level. To this end we generated and analyzed 14 novel draft genomes for Mycoavidus living within the hyphae of Mortierellomycotina fungi. We found that our novel Mycoavidus genomes were significantly reduced compared to free-living Burkholderiales relatives. Using a genome-scale phylogenetic approach including the novel and available existing genomes of Mycoavidus, we show that the genus is an assemblage composed of two independently derived lineages including three well supported clades of Mycoavidus. Using a comparative genomic approach, we shed light on the functional implications of genome reduction, documenting shared and unique gene loss patterns between the three Mycoavidus clades. We found that many endosymbiont isolates demonstrate patterns of vertical transmission and host-specificity, but others are present in phylogenetically disparate hosts. We discuss how reductive evolution and host specificity reflect convergent adaptation to the intrahyphal selective landscape, and commonalities of eukaryotic endosymbiont genome evolution.

Anti-fungal bioactive terpenoids in the bioenergy crop switchgrass (Panicum virgatum) may contribute to ecotype-specific microbiome composition.

Plant derived bioactive small molecules have attracted attention of scientists across fundamental and applied scientific disciplines. We seek to understand the influence of these phytochemicals on rhizosphere and root-associated fungi. We hypothesize that - consistent with accumulating evidence that switchgrass genotype impacts microbiome assembly - differential terpenoid accumulation contributes to switchgrass ecotype-specific microbiome composition. An initial in vitro Petri plate-based disc diffusion screen of 18 switchgrass root derived fungal isolates revealed differential responses to upland- and lowland-isolated metabolites. To identify specific fungal growth-modulating metabolites, we tested fractions from root extracts on three ecologically important fungal isolates - Linnemania elongata, Trichoderma sp. and Fusarium sp. Saponins and diterpenoids were identified as the most prominent antifungal metabolites. Finally, analysis of liquid chromatography-purified terpenoids revealed fungal inhibition structure - activity relationships (SAR). Saponin antifungal activity was primarily determined by the number of sugar moieties - saponins glycosylated at a single core position were inhibitory whereas saponins glycosylated at two core positions were inactive. Saponin core hydroxylation and acetylation were also associated with reduced activity. Diterpenoid activity required the presence of an intact furan ring for strong fungal growth inhibition. These results inform future breeding and biotechnology strategies for crop protection with reduced pesticide application.

The endohyphal microbiome: current progress and challenges for scaling down integrative multi-omic microbiome research.

As microbiome research has progressed, it has become clear that most, if not all, eukaryotic organisms are hosts to microbiomes composed of prokaryotes, other eukaryotes, and viruses. Fungi have only recently been considered holobionts with their own microbiomes, as filamentous fungi have been found to harbor bacteria (including cyanobacteria), mycoviruses, other fungi, and whole algal cells within their hyphae. Constituents of this complex endohyphal microbiome have been interrogated using multi-omic approaches. However, a lack of tools, techniques, and standardization for integrative multi-omics for small-scale microbiomes (e.g., intracellular microbiomes) has limited progress towards investigating and understanding the total diversity of the endohyphal microbiome and its functional impacts on fungal hosts. Understanding microbiome impacts on fungal hosts will advance explorations of how "microbiomes within microbiomes" affect broader microbial community dynamics and ecological functions. Progress to date as well as ongoing challenges of performing integrative multi-omics on the endohyphal microbiome is discussed herein. Addressing the challenges associated with the sample extraction, sample preparation, multi-omic data generation, and multi-omic data analysis and integration will help advance current knowledge of the endohyphal microbiome and provide a road map for shrinking microbiome investigations to smaller scales. Video Abstract.

The microbiome structure of decomposing plant leaves in soil depends on plant species, soil pore sizes, and soil moisture content.

Microbial communities are known as the primary decomposers of all the carbon accumulated in the soil. However, how important soil structure and its conventional or organic management, moisture content, and how different plant species impact this process are less understood. To answer these questions, we generated a soil microcosm with decomposing corn and soy leaves, as well as soil adjacent to the leaves, and compared it to control samples. We then used high-throughput amplicon sequencing of the ITS and 16S rDNA regions to characterize these microbiomes. Leaf microbiomes were the least diverse and the most even in terms of OTU richness and abundance compared to near soil and far soil, especially in their bacterial component. Microbial composition was significantly and primarily affected by niche (leaves vs. soil) but also by soil management type and plant species in the fungal microbiome, while moisture content and pore sizes were more important drivers for the bacterial communities. The pore size effect was significantly dependent on moisture content, but only in the organic management type. Overall, our results refine our understanding of the decomposition of carbon residues in the soil and the factors that influence it, which are key for environmental sustainability and for evaluating changes in ecosystem functions.

Sequencing the Genomes of the First Terrestrial Fungal Lineages: What Have We Learned?

The first genome sequenced of a eukaryotic organism was for Saccharomyces cerevisiae, as reported in 1996, but it was more than 10 years before any of the zygomycete fungi, which are the early-diverging terrestrial fungi currently placed in the phyla Mucoromycota and Zoopagomycota, were sequenced. The genome for Rhizopus delemar was completed in 2008; currently, more than 1000 zygomycete genomes have been sequenced. Genomic data from these early-diverging terrestrial fungi revealed deep phylogenetic separation of the two major clades-primarily plant-associated saprotrophic and mycorrhizal Mucoromycota versus the primarily mycoparasitic or animal-associated parasites and commensals in the Zoopagomycota. Genomic studies provide many valuable insights into how these fungi evolved in response to the challenges of living on land, including adaptations to sensing light and gravity, development of hyphal growth, and co-existence with the first terrestrial plants. Genome sequence data have facilitated studies of genome architecture, including a history of genome duplications and horizontal gene transfer events, distribution and organization of mating type loci, rDNA genes and transposable elements, methylation processes, and genes useful for various industrial applications. Pathogenicity genes and specialized secondary metabolites have also been detected in soil saprobes and pathogenic fungi. Novel endosymbiotic bacteria and viruses have been discovered during several zygomycete genome projects. Overall, genomic information has helped to resolve a plethora of research questions, from the placement of zygomycetes on the evolutionary tree of life and in natural ecosystems, to the applied biotechnological and medical questions.

Interannual dynamics of Tuber melanosporum and fungal communities in productive black truffle orchards amended with truffle nests.

Truffle growers devote great efforts to improve black truffle productivity, developing agronomic practices such as 'truffle nests' (peat amendments that are supplemented with truffle spore inoculum). It has been hypothesized that improved fruiting associated with nests is linked to stimulation of truffle mycelia previously established in soil or to changes generated in soil fungal community. To assess this, we used real-time PCR to quantify black truffle extraradical mycelium during 2 years after nests installation. We also characterized the fungal community via high-throughput amplicon sequencing of the ITS region of rRNA genes. We found that neither the abundance of truffle mycelium in nests nor in the soil-nest interphase was higher than in the bulk soil, which indicates that nests do not improve mycelial growth. The fungal community in nests showed lower richness and Shannon index and was compositionally different from that of soil, which suggests that nests may act as an open niche for fungal colonization that facilitates truffle fruiting. The ectomycorrhizal fungal community showed lower richness in nests. However, no negative relationships between amount of truffle mycelium and reads of other ectomycorrhizal fungi were found, thus countering the hypothesis that ectomycorrhizal competition plays a role in the nest effect.

Regional biogeography versus intra-annual dynamics of the root and soil microbiome.

BACKGROUND: Root and soil microbial communities constitute the below-ground plant microbiome, are drivers of nutrient cycling, and affect plant productivity. However, our understanding of their spatiotemporal patterns is confounded by exogenous factors that covary spatially, such as changes in host plant species, climate, and edaphic factors. These spatiotemporal patterns likely differ across microbiome domains (bacteria and fungi) and niches (root vs. soil). RESULTS: To capture spatial patterns at a regional scale, we sampled the below-ground microbiome of switchgrass monocultures of five sites spanning > 3 degrees of latitude within the Great Lakes region. To capture temporal patterns, we sampled the below-ground microbiome across the growing season within a single site. We compared the strength of spatiotemporal factors to nitrogen addition determining the major drivers in our perennial cropping system. All microbial communities were most strongly structured by sampling site, though collection date also had strong effects; in contrast, nitrogen addition had little to no effect on communities. Though all microbial communities were found to have significant spatiotemporal patterns, sampling site and collection date better explained bacterial than fungal community structure, which appeared more defined by stochastic processes. Root communities, especially bacterial, were more temporally structured than soil communities which were more spatially structured, both across and within sampling sites. Finally, we characterized a core set of taxa in the switchgrass microbiome that persists across space and time. These core taxa represented < 6% of total species richness but > 27% of relative abundance, with potential nitrogen fixing bacteria and fungal mutualists dominating the root community and saprotrophs dominating the soil community. CONCLUSIONS: Our results highlight the dynamic variability of plant microbiome composition and assembly across space and time, even within a single variety of a plant species. Root and soil fungal community compositions appeared spatiotemporally paired, while root and soil bacterial communities showed a temporal lag in compositional similarity suggesting active recruitment of soil bacteria into the root niche throughout the growing season. A better understanding of the drivers of these differential responses to space and time may improve our ability to predict microbial community structure and function under novel conditions.

Accidental cultivation of the European truffle Tuber brumale in North American truffle orchards.

Tuber brumale is a European edible truffle species that is often viewed as a contaminant in truffle orchards, as it visually resembles more valuable black truffles such as T. melanosporum, but differs in aroma and flavor and sells for a much lower price. Although T. brumale is not native to or intentionally cultivated in North America, it was reported to have been accidently introduced into British Columbia in 2014 and North Carolina in 2020. However, in winter of 2021, various truffle orchards in eastern North America produced truffles that differed from the anticipated harvest of T. melanosporum. Molecular analysis of these specimens confirmed T. brumale truffle fruiting bodies from ten orchards distributed across six eastern USA states. Phylogenetic analysis of nuclear ribosomal ITS and 28S DNA sequences indicated that all samples belong to the T. brumale A1 haplogroup, the genetic subgroup of T. brumale that is more common in western Europe. This pattern of widespread fruiting of T. brumale in North American truffle orchards is likely the result of T. brumale being introduced in the initial inoculation of trees used as hosts in T. melanosporum truffle cultivation. We review other examples of introduced non-target truffle species and strategies for limiting their impact on truffle cultivation.

Flocculation of oleaginous green algae with Mortierella alpina fungi.

Microalgae are promising sources of valuable bioproducts such as biofuels, food, and nutraceuticals. However, harvesting microalgae is challenging due to their small size and low biomass concentrations. To address this challenge, bio-flocculation of starchless mutants of Chlamydomonas reinhardtii (sta6/sta7) was investigated with Mortierella alpina, an oleaginous fungus with high concentrations of arachidonic acid (ARA). Triacylglycerides (TAG) reached 85 % of total lipids in sta6 and sta7 through a nitrogen regime. Scanning electron microscopy determined cell-wall attachment and extra polymeric substances (EPS) to be responsible for flocculation. An algal-fungal biomass ratio around 1:1 (three membranes) was optimal for bio-flocculation (80-85 % flocculation efficiency in 24 h). Nitrogen-deprived sta6/sta7 were flocculated with strains of M. alpina (NVP17b, NVP47, and NVP153) with aggregates exhibiting fatty acid profiles similar to C. reinhardtii, with ARA (3-10 % of total fatty acids). This study showcases M. alpina as a strong bio-flocculation candidate for microalgae and advances a mechanistic understanding of algal-fungal interaction.

Co-responses of bacterial and fungal communities to fire management treatments in Mediterranean pyrophytic ecosystems.

Cistus scrublands are pyrophytic ecosystems and occur widely across Mediterranean regions. Management of these scrublands is critical to prevent major disturbances, such as recurring wildfires. This is because management appears to compromise the synergies necessary for forest health and the provision of ecosystem services. Furthermore, it supports high microbial diversity, opening questions of how forest management practices impact belowground associated diversity as research related to this issue is scarce. This study aims to investigate the effects of different fire prevention treatments and site history on bacterial and fungi co-response and co-occurrence patterns over a fire-risky scrubland ecosystem. Two different site histories were studied by applying three different fire prevention treatments and samples were analyzed by amplification and sequencing of ITS2 and 16S rDNA for fungi and bacteria, respectively. The data revealed that site history, especially regarding fire occurrence, strongly influenced the microbial community. Young burnt areas tended to have a more homogeneous and lower microbial diversity, suggesting environmental filtering to a heat-resistant community. In comparison, young clearing history also showed a significant impact on the fungal community but not on the bacteria. Some bacteria genera were efficient predictors of fungal diversity and richness. For instance, Ktedonobacter and Desertibacter were a predictor of the presence of the edible mycorrhizal bolete Boletus edulis. These results demonstrate fungal and bacterial community co-response to fire prevention treatments and provide new tools for forecasting forest management impacts on microbial communities.

Stochastic and deterministic processes shape bioenergy crop microbiomes along a vertical soil niche.

Sustainable biofuel cropping systems aim to address climate change while meeting energy needs. Understanding how soil and plant-associated microbes respond to these different cropping systems is key to promoting agriculture sustainability and evaluating changes in ecosystem functions. Here, we leverage a long-term biofuel cropping system field experiment to dissect soil and root microbiome changes across a soil-depth gradient in poplar, restored prairie and switchgrass to understand their effects on the microbial communities. High throughput amplicon sequencing of the fungal internal transcribed spacer (ITS) and prokaryotic 16S DNA regions showed a common trend of root and soil microbial community richness decreasing and evenness increasing with depth. Ecological niche (root vs. soil) had the strongest effect on community structure, followed by depth, then crop. Stochastic processes dominated the structuring of fungal communities in deeper soil layers while operational taxonomic units (OTUs) in surface soil layers were more likely to co-occur and to be enriched by plant hosts. Prokaryotic communities were dispersal limited at deeper depths. Microbial networks showed a higher density, connectedness, average degree and module size in deeper soils. We observed a decrease in fungal-fungal links and an increase of bacteria-bacteria links with increasing depth in all crops, particularly in the root microbiome.

Investigating Endobacteria that Thrive Within Mucoromycota.

Metagenomics approaches have revealed the importance of Mucoromycota in the evolution and functioning of plant microbiomes. Comprised of three subphyla (Glomeromycotina, Mortierellomycotina, and Mucoromycotina), this early diverging lineage of fungi encompasses species of mycorrhizal fungi, root endophytes, plant pathogens, and many decomposers of plant debris. Interestingly, several taxa of Mucoromycota share a common feature, that is, the presence of endobacteria within their mycelia and spores. The study of these endosymbiotic bacteria is still a challenging task. However, given recent improvements in the sensitivity of culture-free approaches, a deeper understanding of such microbial interactions is now possible and fuels an emerging research field. In this chapter, we report how Mucoromycota, in particular Mortierellomycotina, and their endobacteria can be investigated using a combination of diverse cellular biology, microscopy, and molecular techniques.