Abstract

Abstract

Arbuscular mycorrhizal fungi (AMF, Glomeromycotina), in addition to forming symbioses with the majority of land plants, harbor vertically transmitted endosymbiotic bacteria ‘Candidatus Glomeribacter gigasporarum’ (CaGg) and ‘Candidatus Moeniiplasma glomeromycotorum’ (CaMg). CaGg is a nonessential mutualist of AMF, whereas the lifestyle of CaMg is unknown. To start unraveling the interactions between AMF and their endosymbionts in nature, we examined diversity and distribution of AMF-associated endobacteria in North Atlantic dunes at Cape Cod, MA. Of nearly 500 foredune AMF isolates surveyed during a systematic study, 94% were classified as the Gigasporaceae. 2% of all AMF isolates harbored CaGg, and 88% contained CaMg. CaGg was found only in the Gigasporaceae, whereas CaMg was present in Gigasporaceae, Acaulosporaceae, and Diversisporaceae. Incidence of CaGg across AMF was not affected by any of the environmental parameters measured, whereas distribution of CaMg in one of the hosts was impacted by plant density. CaMg populations associated with AMF individuals displayed high levels of genetic diversity but no evidence of gene flow, suggesting that host physical proximity is not sufficient to facilitate horizontal transmission of CaMg. Lastly, in addition to a novel lineage of CaGg, we discovered Burkholderia-related bacteria previously not known to associate with Glomeromycotina, and likely living inside AMF. They are closely related to free-living Burkholderia and endobacteria of other Mucoromycota fungi. Collectively, we conducted the first ecological study of AMF-associated endobacteria and assessed their diversity and population structure.

Abstract

Mortierellomycotina are excellent living tools to understand the role of Mycoplasma-related endobacteria and the functioning of their interaction with Mucoromycota

Alessandro Desirò¹, Pawel Misztal², Natalie Vande Pol¹, Gian Maria Niccolò Benucci¹, Daniel Jones³, Allen Goldstein², Robert Roberson⁴, Gregory Bonito¹

¹Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI, USA; ²Department of Environmental Science, Policy and Management, University of California Berkeley, Berkeley, CA, USA; ³Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA; ⁴School of Life Sciences, Arizona State University, Tempe, AZ, USA

Emails: ¹adesiro@msu.edu, vandepo7@msu.edu; benucci@msu.edu; bonito@msu.edu; ²pkm@berkeley.edu; ahg@berkeley.edu; ³jonesar4@msu.edu; ⁴robby2@asu.edu;

Fungi can interact with a myriad of organisms, from animals to plants, but also bacteria, other fungi and viruses. Some of these interactions, such as fungal-plant mutualisms, are well-known and have been investigated for a long time. In contrast, only in the last few years has the study of bacterial-fungal interactions blossomed, riding a wave of increasing interest by the scientific community. Bacteria engage in various types of symbiotic associations with fungi, ranging from cooperation to antagonism. These symbioses occur at different levels, with bacteria living inside fungal cells representing the most intimate interaction. Bacterial endosymbionts can be widely found in early-diverging fungi of Mucoromycota, among them Burkholderia-related (BRE) and Mycoplasma-related endobacteria (MRE). BRE represent the best-studied fungal endobacteria and show behavioral shifts from mutualist to weak pathogens. On the contrary, the knowledge on MRE lifestyle is much more limited. Here, we report about the existence of a new bacterial-fungal symbiosis that involves MRE and Mortierellomycotina fungi. We carried out a large-scale screening of hundreds of Mortierellomycotina strains searching for MRE. We used a combination of microscopy, molecular phylogeny, next-generation sequencing and qPCR. We detected MRE within the mycelium of Mortierellomycotina fungi and their presence demonstrates that MRE distribute across the whole Mucoromycota phylum, where they may have lived in the common ancestor. We cleared MRE from their fungal hosts, obtaining a unique experimental system whereby pairs of isogenic fungal lines, with and without MRE, can be employed for comparative functional studies. Cured lines devoid of MRE showed fast growth and improved biomass production. Our data demonstrate that the fungal host experiences some fitness costs in accommodating its endosymbionts and, therefore, provides the first functional insight into the lifestyle of MRE. Our findings suggest that MRE may be antagonistic to their hosts and adapted to a non-lethal parasitic lifestyle in the fungal mycelium. Additional measurements aimed at exploring chemotypic and metabolomic variations across different fungal pairs are expected to provide novel insights into the ecological and evolutionary role of MRE within fungi. Mortierellomycotina offer a unique opportunity to expand the knowledge on MRE and pave the way for potential applications for controlling and using Mucoromycota in agriculture and industry.

Abstract

Interactions between bacteria and fungi have likely evolved during the 600 million years history of terrestrial fungi. This has resulted in bacteria and fungi coexisting in various modern microhabitats and establishing interactions ranging from mutualism to antagonism or neutral coexistence. Such interactions can directly or indirectly impact higher trophic levels, as well as nutrient cycling. Moreover, the interaction between fungal-bacterial interacting partners is dynamic and can rapidly change in response to changes in environmental factors. The same is true for interactions occurring at the cellular level. Bacteria and fungi in close physical contact show relationships ranging from random coexistence to nearly total physiological interdependency. The most intimate relationship yet described consists in bacteria colonizing inner hyphae (endobacteria). In this study, we have investigated the diversity of both endobacteria and bacteria firmly attached to hyphae in fungal strains collections (c.a. 130 fungal strains). Amplicon sequencing of the 16S rRNA gene was used to identify bacterial species in DNA extracted from individual fungal cultures. We have discovered that endobacteria are much more frequent than previously assumed. Moreover, they seem to appear equally distributed in the phyla Basidiomycota, Ascomycota and Zygomycota, and also occur in the distinct phylogenetic lineage of the eukaryotic fungus-like Oomycota. In addition to this, we have started to investigate the rules underpinning this close association. In particular, under environmental conditions affecting negatively the fitness of the fungal host (e.g. temperature, biocides, and poor nutrient supply), we have observed for several fungal models that this tight relation turns to a loose coexistence. Defining the conditions triggering changes in the type of interaction between both partners are key to understand the dynamics of bacterial-fungal interactions. Such a discovery is essential for a better definition of the general mechanisms behind these interactions and their role in microbial ecosystem functioning.

Abstract

The decomposition of dead wood and the resulting nutrient recycling depend on the activity of fungi that has evolved unique enzymatic toolkits. There is also a rich community of bacteria in decaying wood with tentative significant functions. It is well known that forest management practices are important for the maintenance of the diversity of wood decomposing fungi: leaving substrate has a positive effect, whereas forest fragmentation has negative impacts. In this study, we assessed the fungal and bacterial community in downed Picea abies logs in differentially managed forests across environmental gradients in order to identify the major factors structuring these communities. A total of 270 sawdust samples from the interior of 45 P. abies logs were sampled from 3 different landscapes in southern Norway. Within each landscape a cultured forest, a mature or old forest with absence of our red-listed polyporoid focal species, as well as an old growth forest with the presence of our focal species were included. The samples were meta-barcoded by sequencing ITS2 and 16S amplicons of the samples on the Illumina MiSeq platform. Key environmental variables were collected to infer the drivers of the bacterial and fungal communities using ordination methods. We observed a strong biogeographic structuring of the fungal communities and a weaker structuring of the bacteria, while forest management apparently had more limited effects. The co-occurrence pattern of certain taxa of bacteria and fungi were mapped and inferred to reveal potential interactions between them.

Abstract

Fungi are ubiquitous inhabitants of plant tissues and are major drivers of plant and ecosystem health through symbiotic and saprotrophic contributions to nutrient cycles. Interactions between bacteria and fungi can have drastic impacts on fungal phenotypes such as growth, metabolism, and development and thus may alter plant-fungal interaction dynamics. Bacteria that inhabit intracellular space of fungi (endohyphal bacteria, or EHB) can influence fungal phenotypes relevant to interactions with plant tissues. In some cases these symbioses are facultative and the bacteria are transferable, providing new opportunities to compare the genetic and chemical interactions of EHB and their fungal partners in isolation, in natural associations, and in novel partnerships. We evaluated chemical and biological traits of facultative EHB and their impacts on fungal development, metabolism, and transcriptional dynamics. Using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) we assessed the impact of diverse EHB on a focal fungal endophyte, Pestalotiopsis sp. 9143 (P9143). Comparative metabolic analyses showed that association with EHB induced a unique metabolic profile. The native EHB of P9143, Luteibacter mycovicinus, induced a unique metabolic profile containing an analog of the antifungal compound, pestalotether. To evaluate impacts of EHB on fungal gene expression we carried out culture-based phenotypic studies and conducted RNA-seq studies with the Illumina HiSeq platform. We found evidence for a significant alteration in the fungal transcriptional profile and confirm that several primary and secondary metabolic genes are differentially regulated when P9143 and L. mycovicinus are grown in association vs. in isolation. These chemical and phenotypic shifts are potentially important to a competitive lifestyle in plant tissues and illuminate how intimate microbial symbioses may drive polymicrobial interactions. Ongoing work is aimed at analyzing which bacterial genes contribute to the endohyphal lifestyle and elucidating their role in modulating fungal phenotypes and metabolism, especially those that may influence interactions with plant hosts or co-occurring fungi in leaf tissue.