Publications by authors named "Gregory Bonito"

57 Publications

Resolving the Mortierellaceae phylogeny through synthesis of multi-gene phylogenetics and phylogenomics.

Fungal Divers 2020 Sep 16;104(1):267-289. Epub 2020 Sep 16.

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing MI 48824, USA.

Early efforts to classify Mortierellaceae were based on macro- and micromorphology, but sequencing and phylogenetic studies with ribosomal DNA (rDNA) markers have demonstrated conflicting taxonomic groupings and polyphyletic genera. Although some taxonomic confusion in the family has been clarified, rDNA data alone is unable to resolve higher level phylogenetic relationships within Mortierellaceae. In this study, we applied two parallel approaches to resolve the Mortierellaceae phylogeny: low coverage genome (LCG) sequencing and high-throughput, multiplexed targeted amplicon sequencing to generate sequence data for multi-gene phylogenetics. We then combined our datasets to provide a well-supported genome-based phylogeny having broad sampling depth from the amplicon dataset. Resolving the Mortierellaceae phylogeny into monophyletic groups led to the definition of 14 genera, 7 of which are newly proposed. Low-coverage genome sequencing proved to be a relatively cost-effective means of generating a well-resolved phylogeny. The multi-gene phylogenetics approach enabled much greater sampling depth and breadth than the LCG approach, but was unable to resolve higher-level organization of groups. We present this work to resolve some of the taxonomic confusion and provide a genus-level framework to empower future studies on Mortierellaceae diversity, biology, and evolution.
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http://dx.doi.org/10.1007/s13225-020-00455-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7751987PMC
September 2020

, a new edible bolete from China, is associated with insects and plants.

Mycologia 2021 Jan-Feb;113(1):33-42. Epub 2020 Dec 18.

Panzhihua City Academy of Agricultural and Forest Sciences , Panzhihua, Sichuan, China.

is described as new based on collections from southwest China. Phylogenetic analyses of nuclear rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) and portions of nuclear 28S rDNA (28S), translation elongation factor 1-alpha (), and the largest and second largest subunits of RNA polymerase II () support as a novel species in the genus (Boletinellaceae, Boletales). The new species resembles but differs from it in that mature basidiomata have a bright rose-red-colored stipe and a radiate tubular hymenophore with nested pores. Despite extensive searching, has only been found at four sites within a 24-hectare orchard dominated by , which is agriculturally important given its fruit production (loquats). Therefore, this species appears to be endemic and geographically restricted. The ecology of this bolete is also unique. In line with the trophic behavior of other species in the Boletinellaceae, our observations indicate that forms a symbiotic association with the scale insect , identified through sequence analysis of its mitochondrial cytochrome oxidase subunit I () region, to form fungus-insect galls that develop on roots of trees. is an edible mushroom species and is collected from the type location by farmers and sold commercially in limited quantities at local markets alongside and other fungi.
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http://dx.doi.org/10.1080/00275514.2020.1816781DOI Listing
December 2020

Genome Sequencing of Provides Insights into Its Phylogenetic Placement and Mycoparasitism Mechanisms on Morel Mushrooms.

Pathogens 2020 Oct 13;9(10). Epub 2020 Oct 13.

Engineering Research Center of Edible and Medicinal Fungi, Ministry of Education, Jilin Agricultural University, Changchun 130118, China.

Morels ( spp.) are popular edible fungi with significant economic and scientific value. However, white mold disease, caused by , can reduce morel yield by up to 80% in the main cultivation area in China. is a polyphyletic genus and the exact phylogenetic placement of is currently still unclear. Here, we obtained the first high-quality genome sequence of generated through the single-molecule real-time (SMRT) sequencing platform. The assembled draft genome of was 40.2 Mb, had an N50 value of 2.6 Mb and encoded 9454 genes. Phylogenetic analysis of single-copy orthologous genes revealed that is in Hypocreales and closely related to Hypocreaceae, which includes several genera exhibiting a mycoparasitic lifestyle. CAZymes analysis demonstrated that encodes a large number of fungal cell wall degradation enzymes. We identified many gene clusters involved in the production of secondary metabolites known to exhibit antifungal, antibacterial, or insecticidal activities. We further demonstrated through dual culture assays that secretes certain soluble compounds that are inhibitory to the mycelial growth of . This study provides insights into the correct phylogenetic placement of and the molecular mechanisms that underlie pathogenesis.
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http://dx.doi.org/10.3390/pathogens9100834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7650745PMC
October 2020

Large-scale genome sequencing of mycorrhizal fungi provides insights into the early evolution of symbiotic traits.

Nat Commun 2020 10 12;11(1):5125. Epub 2020 Oct 12.

Université de Lorraine, Institut national de recherche pour l'agriculture, l'alimentation et l' environnement, UMR Interactions Arbres/Microorganismes, Centre INRAE Grand Est-Nancy, 54280, Champenoux, France.

Mycorrhizal fungi are mutualists that play crucial roles in nutrient acquisition in terrestrial ecosystems. Mycorrhizal symbioses arose repeatedly across multiple lineages of Mucoromycotina, Ascomycota, and Basidiomycota. Considerable variation exists in the capacity of mycorrhizal fungi to acquire carbon from soil organic matter. Here, we present a combined analysis of 135 fungal genomes from 73 saprotrophic, endophytic and pathogenic species, and 62 mycorrhizal species, including 29 new mycorrhizal genomes. This study samples ecologically dominant fungal guilds for which there were previously no symbiotic genomes available, including ectomycorrhizal Russulales, Thelephorales and Cantharellales. Our analyses show that transitions from saprotrophy to symbiosis involve (1) widespread losses of degrading enzymes acting on lignin and cellulose, (2) co-option of genes present in saprotrophic ancestors to fulfill new symbiotic functions, (3) diversification of novel, lineage-specific symbiosis-induced genes, (4) proliferation of transposable elements and (5) divergent genetic innovations underlying the convergent origins of the ectomycorrhizal guild.
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http://dx.doi.org/10.1038/s41467-020-18795-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7550596PMC
October 2020

Patient propagules: Do soil archives preserve the legacy of fungal and prokaryotic communities?

PLoS One 2020 11;15(8):e0237368. Epub 2020 Aug 11.

Plant, Soil and Microbial Science Department, Michigan State University, East Lansing, MI, United States of America.

Soil archives are an important resource in agronomic and ecosystem sciences. If microbial communities could be reconstructed from archived soil DNA, as prehistoric plant communities are reconstructed via pollen data, soil archive resources would assume even greater value for reconstructing land-use history, forensic science, and biosphere modelling. Yet, the effects of long-term soil archival on the preservation of microbial DNA is still largely unknown. To address this, we assessed the capacity of high-throughput sequencing (Illumina MiSeq) of ITS (internal transcribed spacer) and prokaryotic 16S rRNA genes for reconstructing soil microbial communities across a 20 years time-series. We studied air-dried soil archives and fresh soil samples taken from Populus bioenergy and deciduous forest research plots at the Kellogg Biological Station. Habitat and archival time explained significant amounts of variation in soil microbial α- and β-diversity both in fungal and prokaryotic communities. We found that microbial richness, diversity, and abundance generally decreased with storage time, but varied between habitat and taxonomic groups. The high relative abundance of ectomycorrhizal species including Hebeloma and Cortinarius detected in older soil archives raises questions regarding traits such as long-term persistence and viability of ectomycorrhizal propagules in soils, with relevance to forest health and ecosystem succession. Talaromyces, Paecilomyces and Epicoccum spp. were detected in fresh and across 20-year-old archived soils and were also cultured from these soils demonstrating their long-term spore viability. In summary, we found that microbial DNA in air-dried soils archived over the past 20 years degraded with time, in a manner that differed between soil types and phylogenetic groups of microbes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0237368PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418970PMC
October 2020

Crop Management Impacts the Soybean () Microbiome.

Front Microbiol 2020 3;11:1116. Epub 2020 Jun 3.

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, United States.

Soybean () is an important leguminous crop that is grown throughout the United States and around the world. In 2016, soybean was valued at $41 billion USD in the United States alone. Increasingly, soybean farmers are adopting alternative management strategies to improve the sustainability and profitability of their crop. Various benefits have been demonstrated for alternative management systems, but their effects on soybean-associated microbial communities are not well-understood. In order to better understand the impact of crop management systems on the soybean-associated microbiome, we employed DNA amplicon sequencing of the Internal Transcribed Spacer (ITS) region and 16S rRNA genes to analyze fungal and prokaryotic communities associated with soil, roots, stems, and leaves. Soybean plants were sampled from replicated fields under long-term conventional, no-till, and organic management systems at three time points throughout the growing season. Results indicated that sample origin was the main driver of beta diversity in soybean-associated microbial communities, but management regime and plant growth stage were also significant factors. Similarly, differences in alpha diversity are driven by compartment and sample origin. Overall, the organic management system had lower fungal and bacterial Shannon diversity. In prokaryotic communities, aboveground tissues were dominated by and while belowground samples were dominated by and Aboveground fungal communities were dominated by across all management systems, while belowground samples were dominated by and . Specific taxa including potential plant beneficials such as were indicator species of the conventional and organic management systems. No-till management increased the abundance of groups known to contain plant beneficial organisms such as and Glomeromycotina. Network analyses show different highly connected hub taxa were present in each management system. Overall, this research demonstrates how specific long-term cropping management systems alter microbial communities and how those communities change throughout the growth of soybean.
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http://dx.doi.org/10.3389/fmicb.2020.01116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283522PMC
June 2020

Evidence for Co-evolutionary History of Early Diverging Lycopodiaceae Plants With Fungi.

Front Microbiol 2019 15;10:2944. Epub 2020 Jan 15.

School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.

Lycopods are tracheophytes in the Kingdom Plantae and represent one of the oldest lineages of living vascular plants. Symbiotic interactions between these plants with fungi and bacteria, including fine root endophytes in Endogonales, have been hypothesized to have helped early diverging plant lineages colonize land. However, attempts to study the lycopod rhizobiome in its natural environment are still limited. In this study, we used Illumina amplicon sequencing to characterize fungal and bacterial diversity in nine Lycopodiaceae (club moss) species collected in New Zealand. This was done with generic fungal ITS rDNA primers, as well as Endogonales- and arbuscular mycorrhizal fungi (AMF)-selective primer sets targeting the 18S rDNA, and generic bacterial primers targeting the V4 region of the 16S rDNA. We found that the Lycopodiaceae rhizobiome was comprised of an unexpected high frequency of Basidiomycota and Ascomycota coincident with a low abundance of Endogonales and Glomerales. The distribution and abundance of Endogonales varied with host lycopod, and included a novel taxon as well as a single operational taxonomic unit (OTU) that was detected across all plant species. The Lycopodiaceae species with the greatest number and also most unique OTUs was , while the plant species that shared the most fungal OTUs were and . The bacterial OTU distribution was generally not consistent with fungal OTU distribution. For example, community dissimilarity analysis revealed strong concordance between the evolutionary histories of host plants with the fungal community but not with the bacterial community, indicating that Lycopodiaceae have evolved specific relationships with their fungal symbionts. Notably, nearly 16% of the ITS rDNA fungal diversity detected in the Lycopodiaceae rhizobiome remained poorly classified, indicating there is much plant-associated fungal diversity left to describe in New Zealand.
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http://dx.doi.org/10.3389/fmicb.2019.02944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6974469PMC
January 2020

Draft Genome Sequence of the Ectomycorrhizal Ascomycete .

Microbiol Resour Announc 2019 Dec 12;8(50). Epub 2019 Dec 12.

Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, Michigan, USA.

is a pioneer ectomycorrhizal fungus with facultative saprophytic capacities. Here, we sequenced the genome of strain Sb_GMNB300, which is estimated at 51.6 Mb in size with 872 assembled contigs accounting for 12,597 predicted coding genes. This genome will be useful for comparative studies of Pezizales ectomycorrhizal symbioses.
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http://dx.doi.org/10.1128/MRA.00857-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6908789PMC
December 2019

Microfluidics and Metabolomics Reveal Symbiotic Bacterial-Fungal Interactions Between and Include Metabolite Exchange.

Front Microbiol 2019 1;10:2163. Epub 2019 Oct 1.

Department of Biology, Duke University, Durham, NC, United States.

We identified two poplar ( sp.)-associated microbes, the fungus, strain AG77, and the bacterium, strain BT03, that mutually promote each other's growth. Using culture assays in concert with a novel microfluidic device to generate time-lapse videos, we found growth specific media differing in pH and pre-conditioned by microbial growth led to increased fungal and bacterial growth rates. Coupling microfluidics and comparative metabolomics data results indicated that observed microbial growth stimulation involves metabolic exchange during two ordered events. The first is an emission of fungal metabolites, including organic acids used or modified by bacteria. A second signal of unknown nature is produced by bacteria which increases fungal growth rates. We find this symbiosis is initiated in part by metabolic exchange involving fungal organic acids.
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http://dx.doi.org/10.3389/fmicb.2019.02163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779839PMC
October 2019

Fungal and bacterial community dynamics in substrates during the cultivation of morels (Morchella rufobrunnea) indoors.

FEMS Microbiol Lett 2019 09;366(17)

Department of Microbiology and Molecular Genetics, Michigan State University, 567 Wilson Rd, East Lansing MI, 48823, USA.

Morel mushrooms (Morchella, Pezizales) are highly prized edible fungi. Approaches to cultivate morels indoors in pasteurized composted substrates have been successful for Morchella rufobrunnea. We used DNA amplicon sequencing of the Internal Transcribed Spacer (ITS) ribosomal DNA and 16S rRNA gene to follow bacterial and fungal communities in substrates during indoor morel cultivation. Our goal was to determine changes in microbial communities at key stages of morel cultivation, which included primordia development, fundament initiation, differentiation and maturation. Additionally, we compared microbial communities between trays that successfully fruited to those that produced conidia and primordia but aborted before ascocarp formation (non-fruiting). The prokaryotic community was dominated by Firmicutes belonging to Bacillus and Paenibacillus with a lower abundance of Flavobacteria. At earlier stages, the fungal community was dominated by Pezizomycetes including Morchella and other species, whereas, later in the cropping cycle Sordariomycetes dominated. Additionally, differences were observed between trays with successful fruiting, which were dominated by Gilmaniella; compared to trays that did not fruit, which were dominated by Cephalotrichum. Our findings inform understanding of microbial community dynamics during morel cultivation, and show that fungal genera, such as Gilmaniella, and prokaryotic genera, such as Bacillus, are abundant in substrates that support M. rufobrunnea fruiting.
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http://dx.doi.org/10.1093/femsle/fnz215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6836762PMC
September 2019

Microbial communities associated with the black morel cultivated in greenhouses.

PeerJ 2019 26;7:e7744. Epub 2019 Sep 26.

CAS Key Laboratory for East Asia Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Yunnan, China.

Morels ( spp.) are iconic edible mushrooms with a long history of human consumption. Some microbial taxa are hypothesized to be important in triggering the formation of morel primordia and development of fruiting bodies, thus, there is interest in the microbial ecology of these fungi. To identify and compare fungal and prokaryotic communities in soils where is cultivated in outdoor greenhouses, ITS and 16S rDNA high throughput amplicon sequencing and microbiome analyses were performed. , , , and were found to comprise the core microbiome of ascocarps. These bacterial taxa were also abundant in the soil beneath growing fruiting bodies. A total of 29 bacterial taxa were found to be statistically associated to fruiting bodies. Bacterial community network analysis revealed high modularity with some 16S rDNA operational taxonomic unit clusters living in specialized fungal niches (e.g., pileus, stipe). Other fungi dominating the soil mycobiome beneath morels included , , and . This research informs understanding of microbial indicators and potential facilitators of ecology and fruiting body production.
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http://dx.doi.org/10.7717/peerj.7744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6766373PMC
September 2019

Algal-fungal symbiosis leads to photosynthetic mycelium.

Elife 2019 07 16;8. Epub 2019 Jul 16.

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, United States.

Mutualistic interactions between free-living algae and fungi are widespread in nature and are hypothesized to have facilitated the evolution of land plants and lichens. In all known algal-fungal mutualisms, including lichens, algal cells remain external to fungal cells. Here, we report on an algal-fungal interaction in which algal cells become internalized within the hyphae of the fungus . This apparent symbiosis begins with close physical contact and nutrient exchange, including carbon and nitrogen transfer between fungal and algal cells as demonstrated by isotope tracer experiments. This mutualism appears to be stable, as both partners remain physiologically active over months of co-cultivation, leading to the eventual internalization of photosynthetic algal cells, which persist to function, grow and divide within fungal hyphae. and are biotechnologically important species for lipids and biofuel production, with available genomes and molecular tool kits. Based on the current observations, they provide unique opportunities for studying fungal-algal mutualisms including mechanisms leading to endosymbiosis.
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http://dx.doi.org/10.7554/eLife.47815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6634985PMC
July 2019

Increasing access to microfluidics for studying fungi and other branched biological structures.

Fungal Biol Biotechnol 2019 10;6. Epub 2019 Jun 10.

1Biosciences Division, Oak Ridge National Laboratory, PO Box 2008, MS 6445, Oak Ridge, TN 37831 USA.

Background: Microfluidic systems are well-suited for studying mixed biological communities for improving industrial processes of fermentation, biofuel production, and pharmaceutical production. The results of which have the potential to resolve the underlying mechanisms of growth and transport in these complex branched living systems. Microfluidics provide controlled environments and improved optical access for real-time and high-resolution imaging studies that allow high-content and quantitative analyses. Studying growing branched structures and the dynamics of cellular interactions with both biotic and abiotic cues provides context for molecule production and genetic manipulations. To make progress in this arena, technical and logistical barriers must be overcome to more effectively deploy microfluidics in biological disciplines. A principle technical barrier is the process of assembling, sterilizing, and hydrating the microfluidic system; the lack of the necessary equipment for the preparatory process is a contributing factor to this barrier. To improve access to microfluidic systems, we present the development, characterization, and implementation of a microfluidics assembly and packaging process that builds on self-priming point-of-care principles to achieve "ready-to-use microfluidics."

Results: We present results from domestic and international collaborations using novel microfluidic architectures prepared with a unique packaging protocol. We implement this approach by focusing primarily on filamentous fungi; we also demonstrate the utility of this approach for collaborations on plants and neurons. In this work we (1) determine the shelf-life of ready-to-use microfluidics, (2) demonstrate biofilm-like colonization on fungi, (3) describe bacterial motility on fungal hyphae (fungal highway), (4) report material-dependent bacterial-fungal colonization, (5) demonstrate germination of vacuum-sealed seeds in microfluidics stored for up to 2 weeks, and (6) observe bidirectional cytoplasmic streaming in fungi.

Conclusions: This pre-packaging approach provides a simple, one step process to initiate microfluidics in any setting for fungal studies, bacteria-fungal interactions, and other biological inquiries. This process improves access to microfluidics for controlling biological microenvironments, and further enabling visual and quantitative analysis of fungal cultures.
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http://dx.doi.org/10.1186/s40694-019-0071-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6556955PMC
June 2019

Fungal-Bacterial Networks in the Rhizobiome Are Impacted by Soil Properties and Host Genotype.

Front Microbiol 2019 29;10:481. Epub 2019 Mar 29.

Department of Biology, Duke University, Durham, NC, United States.

Plant root-associated microbial symbionts comprise the plant rhizobiome. These microbes function in provisioning nutrients and water to their hosts, impacting plant health and disease. The plant microbiome is shaped by plant species, plant genotype, soil and environmental conditions, but the contributions of these variables are hard to disentangle from each other in natural systems. We used bioassay common garden experiments to decouple plant genotype and soil property impacts on fungal and bacterial community structure in the rhizobiome. High throughput amplification and sequencing of 16S, ITS, 28S and 18S rDNA was accomplished through 454 pyrosequencing. Co-association patterns of fungal and bacterial taxa were assessed with 16S and ITS datasets. Community bipartite fungal-bacterial networks and PERMANOVA results attribute significant difference in fungal or bacterial communities to soil origin, soil chemical properties and plant genotype. Indicator species analysis identified a common set of root bacteria as well as endophytic and ectomycorrhizal fungi associated with in different soils. However, no single taxon, or consortium of microbes, was indicative of a particular genotype. Fungal-bacterial networks were over-represented in arbuscular mycorrhizal, endophytic, and ectomycorrhizal fungi, as well as bacteria belonging to the orders Rhizobiales, Chitinophagales, Cytophagales, and Burkholderiales. These results demonstrate the importance of soil and plant genotype on fungal-bacterial networks in the belowground plant microbiome.
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http://dx.doi.org/10.3389/fmicb.2019.00481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450171PMC
March 2019

Genome Sequencing Illustrates the Genetic Basis of the Pharmacological Properties of .

Genes (Basel) 2019 03 1;10(3). Epub 2019 Mar 1.

Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural University, Changchun 130118, China.

is a precious edible mushroom that is widely grown in Asia and known for its useful medicinal properties. Here, we present a high-quality genome of using the single-molecule real-time (SMRT) sequencing platform. The genome, which is the first complete genome to be sequenced in the family , was 38.67 Mbp, with an N50 of 3.5 Mbp, encoding 15,251 proteins. Based on our phylogenetic analysis, the diverged ~174 million years ago. Several genes and gene clusters associated with lignocellulose degradation, secondary metabolites, and polysaccharide biosynthesis were identified in , and compared with other medicinal mushrooms. In particular, we identified two terpenoid-associated gene clusters, each containing a gene encoding a sesterterpenoid synthase adjacent to a gene encoding a cytochrome P450 enzyme. These clusters might participate in the biosynthesis of incarnal, a known bioactive sesterterpenoid produced by . Through a transcriptomic analysis comparing the mycelium and fruiting body, we also demonstrated that the genes associated with terpenoid biosynthesis were generally upregulated in the mycelium, while those associated with polysaccharide biosynthesis were generally upregulated in the fruiting body. This study provides insights into the genetic basis of the medicinal properties of laying a framework for future characterization of bioactive proteins and pharmaceutical uses of this fungus.
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http://dx.doi.org/10.3390/genes10030188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6470497PMC
March 2019

Fungal Endophytes of Alter Host Phenotype, Gene Expression, and Rhizobiome Composition.

Mol Plant Microbe Interact 2019 Jul 10;32(7):853-864. Epub 2019 Jun 10.

3 Department of Biology, Duke University, Durham, NC, U.S.A.

and genera include common species of soil fungi that are frequently detected as root endophytes in many plants, including spp. However, the ecological roles of these and other endophytic fungi with respect to plant growth and function are still not well understood. The functional ecology of two key taxa from the rhizobiome, PMI93 and PMI82, was studied by coupling forest soil bioassays with environmental metatranscriptomics. Using soil bioassay experiments amended with fungal inoculants, was observed to promote the growth of . This response was cultivar independent. In contrast, had no visible effect on growth. Metatranscriptomic studies revealed that these fungi impacted rhizophytic and endophytic activities in and induced shifts in soil and root microbial communities. Differential expression of core genes in roots was observed in response to both fungal species. Expression of genes for lipid signaling and nutrient uptake were upregulated, and expression of genes associated with gibberellin signaling were altered in plants inoculated with , but not . Upregulation of genes for growth promotion, downregulation of genes for several leucine-rich repeat receptor kinases, and alteration of expression of genes associated with plant defense responses (e.g., jasmonic acid, salicylic acid, and ethylene signal pathways) also suggest that manipulates plant defenses while promoting plant growth.
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http://dx.doi.org/10.1094/MPMI-05-18-0133-RDOI Listing
July 2019

Phylogenomics of Endogonaceae and evolution of mycorrhizas within Mucoromycota.

New Phytol 2019 04 12;222(1):511-525. Epub 2019 Jan 12.

Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR, 97331, USA.

Endogonales (Mucoromycotina), composed of Endogonaceae and Densosporaceae, is the only known non-Dikarya order with ectomycorrhizal members. They also form mycorrhizal-like association with some nonspermatophyte plants. It has been recently proposed that Endogonales were among the earliest mycorrhizal partners with land plants. It remains unknown whether Endogonales possess genomes with mycorrhizal-lifestyle signatures and whether Endogonales originated around the same time as land plants did. We sampled sporocarp tissue from four Endogonaceae collections and performed shotgun genome sequencing. After binning the metagenome data, we assembled and annotated the Endogonaceae genomes. We performed comparative analysis on plant-cell-wall-degrading enzymes (PCWDEs) and small secreted proteins (SSPs). We inferred phylogenetic placement of Endogonaceae and estimated the ages of Endogonaceae and Endogonales with expanded taxon sampling. Endogonaceae have large genomes with high repeat content, low diversity of PCWDEs, but without elevated SSP/secretome ratios. Dating analysis estimated that Endogonaceae originated in the Permian-Triassic boundary and Endogonales originated in the mid-late Silurian. Mycoplasma-related endobacterium sequences were identified in three Endogonaceae genomes. Endogonaceae genomes possess typical signatures of mycorrhizal lifestyle. The early origin of Endogonales suggests that the mycorrhizal association between Endogonales and plants might have played an important role during the colonization of land by plants.
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http://dx.doi.org/10.1111/nph.15613DOI Listing
April 2019

Description and distribution of sp. nov. and in the Transmexican Volcanic Belt.

MycoKeys 2018 11(41):17-27. Epub 2018 Oct 11.

Instituto Tecnológico de Ciudad Victoria. Av. Portes Gil 1301 Poniente, 87010 Ciudad Victoria, Tamaulipas, México.

The genus is a lineage of diverse ectomycorrhizal, hypogeous, sequestrate ascomycete fungi that are native to temperate forests in the Northern Hemisphere. Recently, many new species of have been described in North America and Asia, based on morphological characteristics and molecular data. Here we describe and illustrate a new species, , based upon phylogenetic analysis and morphological description. We also present a new record for in México. These two species are distributed in the Transmexican Volcanic Belt in the states of México, Michoacán, Guanajuato, Querétaro and Tlaxcala at altitudes between 2,000 and 3,200 meters. These species are associated with () and forests ().
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http://dx.doi.org/10.3897/mycokeys.41.28130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6194138PMC
October 2018

Fungal, Bacterial, and Archaeal Diversity in Soils Beneath Native and Introduced Plants in Fiji, South Pacific.

Microb Ecol 2019 Jul 4;78(1):136-146. Epub 2018 Oct 4.

Department of Plants, Soil and Microbial Sciences, Michigan State University, East Lansing, MI, 48824, USA.

The Fiji Islands is an archipelago of more than 330 islands located in the tropics of the South Pacific Ocean. Microbial diversity and biogeography in this region is still not understood. Here, we present the first molecular characterization of fungal, bacterial, and archaeal communities in soils from different habitats within the largest Fijian island, Viti Levu. Soil samples were collected from under native vegetation in maritime-, forest-, stream-, grassland-, and casuarina-dominated habitats, as well as from under the introduced agricultural crops sugarcane, cassava, pine, and mahogany. Soil microbial diversity was analyzed through MiSeq amplicon sequencing of 16S (for prokaryotes), ITS, LSU ribosomal DNA (for fungi). Prokaryotic communities were dominated by Proteobacteria (~ 25%), Acidobacteria (~ 19%), and Actinobacteria (~ 17%), and there were no indicator species associated with particular habitats. ITS and LSU were congruent in β-diversity patterns of fungi, and fungal communities were dominated by Ascomycota (~ 57-64%), followed by Basidiomycota (~ 20-23%) and Mucoromycota (~ 10%) according to ITS, or Chytridiomycota (~ 9%) according to LSU. Indicator species analysis of fungi found statistical associations of Cenococcum, Wilcoxina, and Rhizopogon to Pinus caribaea. We hypothesize these obligate biotrophic fungi were co-introduced with their host plant. Entoloma was statistically associated with grassland soils, and Fusarium and Lecythophora with soils under cassava. Observed richness varied from 65 (casuarina) to 404 OTUs (cassava) for fungi according to ITS region, and from 1268 (pine) to 2931 OTUs (cassava) for bacteria and archaea. A major finding of this research is that nearly 25% of the fungal OTUs are poorly classified, indicative of novel biodiversity in this region. This preliminary survey provides important baseline data on fungal, bacterial, and archaeal diversity and biogeography in the Fiji Islands.
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http://dx.doi.org/10.1007/s00248-018-1266-1DOI Listing
July 2019

Tuber brennemanii and Tuber floridanum: Two new Tuber species are among the most commonly detected ectomycorrhizal taxa within commercial pecan (Carya illinoinensis) orchards.

Mycologia 2018 Jul-Aug;110(4):780-790. Epub 2018 Aug 21.

a Department of Plant Pathology , University of Florida , Gainesville , Florida 32611.

Truffles are sequestrate hypogeous fungi, and most form ectomycorrhizal (ECM) associations with trees. Truffles belonging to the genus Tuber (Pezizales, Ascomycota), "true truffles," associate with diverse plant hosts, including economically important species such as pecan (Carya illinoinensis). Morphological and phylogenetic studies delimited several major lineages of Tuber, which include many cryptic and undescribed species. One of these, the Maculatum clade, is a speciose group characterized by relatively small, light-colored ascomata that have alveolate-reticulate spores. Here, we describe two new species in the Maculatum clade, Tuber brennemanii and T. floridanum (previously identified as Tuber sp. 36 and Tuber sp. 47). We delineate these two species by phylogenetic analyses of nuc ITS1-5.8S-ITS2 (= ITS) and partial 28S rDNA (= LSU), and through morphological analysis. A recent collection of T. floridanum from a pecan orchard in Brazil indicates that this species was introduced there on the roots of pecan seedlings. Systematic studies of ascomata and ECM fungal communities indicate that these species are geographically widespread and common ECM symbionts of pecans and other members of the Fagales, particularly in sites with disturbed soils and nutrient enrichment.
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http://dx.doi.org/10.1080/00275514.2018.1490121DOI Listing
January 2019

Enhancing oil production and harvest by combining the marine alga and the oleaginous fungus .

Biotechnol Biofuels 2018 22;11:174. Epub 2018 Jun 22.

1Department of Energy-Plant Research Laboratory, Michigan State University, East Lansing, MI 48824 USA.

Background: Although microalgal biofuels have potential advantages over conventional fossil fuels, high production costs limit their application in the market. We developed bio-flocculation and incubation methods for the marine alga, CCMP1779, and the oleaginous fungus, AG77, resulting in increased oil productivity.

Results: By growing separately and then combining the cells, the mycelium could efficiently capture due to an intricate cellular interaction between the two species leading to bio-flocculation. Use of a high-salt culture medium induced accumulation of triacylglycerol (TAG) and enhanced the contents of polyunsaturated fatty acids (PUFAs) including arachidonic acid and docosahexaenoic acid in . To increase TAG productivity in the alga, we developed an effective, reduced nitrogen-supply regime based on ammonium in environmental photobioreactors. Under optimized conditions, produced high levels of TAG that could be indirectly monitored by following chlorophyll content. Combining and to initiate bio-flocculation yielded high levels of TAG and total fatty acids, with ~ 15 and 22% of total dry weight (DW), respectively, as well as high levels of PUFAs. Genetic engineering of for higher TAG content in nutrient-replete medium was accomplished by overexpressing , a gene encoding the type II acyl-CoA:diacylglycerol acyltransferase 5. Combined with bio-flocculation, this approach led to increased production of TAG under nutrient-replete conditions (~ 10% of DW) compared to the wild type (~ 6% of DW).

Conclusions: The combined use of and with available genomes and genetic engineering tools for both species opens up new avenues to improve biofuel productivity and allows for the engineering of polyunsaturated fatty acids.
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http://dx.doi.org/10.1186/s13068-018-1172-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013958PMC
June 2018

Emission Factors of Microbial Volatile Organic Compounds from Environmental Bacteria and Fungi.

Environ Sci Technol 2018 08 13;52(15):8272-8282. Epub 2018 Jul 13.

Department of Biology , Duke University , Durham , North Carolina 27708 , United States.

Knowledge of the factors controlling the diverse chemical emissions of common environmental bacteria and fungi is crucial because they are important signal molecules for these microbes that also could influence humans. We show here not only a high diversity of mVOCs but that their abundance can differ greatly in different environmental contexts. Microbial volatiles exhibit dynamic changes across microbial growth phases, resulting in variance of composition and emission rate of species-specific and generic mVOCs. In vitro experiments documented emissions of a wide range of mVOCs (>400 different chemicals) at high time resolution from diverse microbial species grown under different controlled conditions on nutrient media, or residential structural materials ( N = 54, N = 23). Emissions of mVOCs varied not only between microbial taxa at a given condition but also as a function of life stage and substrate type. We quantify emission factors for total and specific mVOCs normalized for respiration rates to account for the microbial activity during their stationary phase. Our VOC measurements of different microbial taxa indicate that a variety of factors beyond temperature and water activity, such as substrate type, microbial symbiosis, growth phase, and lifecycle affect the magnitude and composition of mVOC emission.
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http://dx.doi.org/10.1021/acs.est.8b00806DOI Listing
August 2018

Two new endophytic Atractiellomycetes, Atractidochium hillariae and Proceropycnis hameedii.

Mycologia 2018 Jan-Feb;110(1):136-146

c Department of Ecology, Evolution, and Marine Biology , University of California-Santa Barbara , Santa Barbara , California 93106.

Sterile fungal isolates are often recovered in leaf and root endophytic studies, although these seldom play a significant role in downstream analyses. The authors sought to identify and characterize two such endophytes-one representing the most commonly recovered fungal isolate in recent studies of needle endophytes of Pinus taeda and the other representing a rarely isolated root endophyte of Populus trichocarpa. Both are shown by DNA sequencing to be undescribed species of Atractiellomycetes (Pucciniomycotina, Basidiomycota), a poorly characterized class of mostly plant-associated and presumably saprobic microfungi. The authors describe the new genus and species Atractidochium hillariae (Phleogenaceae) and the new species Proceropycnis hameedii (Hoehnelomycetaceae), both in the Atractiellales, to accommodate these unusual isolates. Following incubations of 1-2 mo, A. hillariae produces minute white sporodochia, similar to those produced by several other members of Atractiellales, whereas Pr. hameedii forms conidia singly or in chains in a manner similar to its sister species Pr. pinicola. Additionally, we provide a taxonomic revision of Atractiellomycetes based on multilocus analyses and propose the new genera Neogloea (Helicogloeaceae) and Bourdotigloea (Phleogenaceae) to accommodate ex-Helicogloea species that are not congeneric with the type H. lagerheimii. Atractiellomycetes consists of a single order, Atractiellales, and three families, Hoehnelomycetaceae, Phleogenaceae, and Helicogloeaceae. Accumulated evidence suggests that Atractiellomycetes species are common but infrequently isolated members of plant foliar and root endobiomes.
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http://dx.doi.org/10.1080/00275514.2018.1446650DOI Listing
December 2018

sp. nov., a truffle species from Mexico belonging to the Maculatum clade (Tuberaceae, Pezizales).

MycoKeys 2018 28(30):61-72. Epub 2018 Feb 28.

US Department of Agriculture, Forest Service, Northern Research Station, 3200 Jefferson Way, Corvallis, Oregon, 97331, USA.

A new species of truffle, , is described from central Mexico. can be distinguished from other related species synoptically by a combination of morphological features including ascospore size, pellis cells with irregular thickness, cystidia, ascoma colour and associated host ( an endemic species from central Mexico); sequence variation on the ITS rDNA also distinguishes from related species. A phylogenetic analysis of the ITS rDNA demonstrates that belongs to the Maculatum clade and is unique from other similar small, white-cream coloured species distributed in north-eastern Mexico such as and .
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http://dx.doi.org/10.3897/mycokeys.30.22887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5904530PMC
February 2018

RNA-based analyses reveal fungal communities structured by a senescence gradient in the moss Dicranum scoparium and the presence of putative multi-trophic fungi.

New Phytol 2018 06 31;218(4):1597-1611. Epub 2018 Mar 31.

Department of Biology, Duke University, Durham, NC, 27708, USA.

Diverse plant-associated fungi are thought to have symbiotrophic and saprotrophic states because they can be isolated from both dead and living plant tissues. However, such tissues often are separated in time and space, and fungal activity at various stages of plant senescence is rarely assessed directly in fungal community studies. We used fungal ribosomal RNA metatranscriptomics to detect active fungal communities across a natural senescence gradient within wild-collected gametophytes of Dicranum scoparium (Bryophyta) to understand the distribution of active fungal communities in adjacent living, senescing and dead tissues. Ascomycota were active in all tissues across the senescence gradient. By contrast, Basidiomycota were prevalent and active in senescing and dead tissues. Several fungi were detected as active in living and dead tissues, suggesting their capacity for multi-trophy. Differences in community assembly detected by metatranscriptomics were echoed by amplicon sequencing of cDNA and compared to culture-based inferences and observation of fungal fruit bodies in the field. The combination of amplicon sequencing of cDNA and metatranscriptomics is promising for studying symbiotic systems with complex microbial diversity, allowing for the simultaneous detection of their presence and activity.
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http://dx.doi.org/10.1111/nph.15092DOI Listing
June 2018

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

ISME J 2018 06 23;12(7):1743-1757. Epub 2018 Feb 23.

Department of Plant Soil and Microbial Sciences, Michigan State University, East Lansing, MI, USA.

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.
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http://dx.doi.org/10.1038/s41396-018-0053-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6018737PMC
June 2018

Bacterial-fungal interactions: ecology, mechanisms and challenges.

FEMS Microbiol Rev 2018 05;42(3):335-352

Helmholtz Centre for Environmental Research-UFZ, Department of Environmental Microbiology, Permoserstraße 15, 04318 Leipzig, Germany.

Fungi and bacteria are found living together in a wide variety of environments. Their interactions are significant drivers of many ecosystem functions and are important for the health of plants and animals. A large number of fungal and bacterial families engage in complex interactions that lead to critical behavioural shifts of the microorganisms ranging from mutualism to antagonism. The importance of bacterial-fungal interactions (BFI) in environmental science, medicine and biotechnology has led to the emergence of a dynamic and multidisciplinary research field that combines highly diverse approaches including molecular biology, genomics, geochemistry, chemical and microbial ecology, biophysics and ecological modelling. In this review, we discuss recent advances that underscore the roles of BFI across relevant habitats and ecosystems. A particular focus is placed on the understanding of BFI within complex microbial communities and in regard of the metaorganism concept. We also discuss recent discoveries that clarify the (molecular) mechanisms involved in bacterial-fungal relationships, and the contribution of new technologies to decipher generic principles of BFI in terms of physical associations and molecular dialogues. Finally, we discuss future directions for research in order to stimulate synergy within the BFI research area and to resolve outstanding questions.
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http://dx.doi.org/10.1093/femsre/fuy008DOI Listing
May 2018

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

IMA Fungus 2017 Dec 3;8(2):245-257. Epub 2017 Oct 3.

Department of Plant Soil and Microbial Sciences, Michigan State University, East Lansing, MI 48824, USA.

is a lineage of early diverging fungi within . 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 , 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 using sporophores collected over the past three decades from four continents. Our results show that harbour significant undescribed diversity and form two deeply divergent and well-supported phylogenetic clades, which we delimit as the families and fam. nov. The family consists of the genus , and many diverse lineages known only from environmental DNA sequences of plant-endosymbiotic fungi. Within there are two clades. One corresponds to and includes the type species, . Species of 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, gen. nov. Members of that genus ( and 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 and will be important for understanding fungal innovations including evolution of macroscopic sporophores and symbioses with plants.
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http://dx.doi.org/10.5598/imafungus.2017.08.02.03DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5729711PMC
December 2017

CONSTAX: a tool for improved taxonomic resolution of environmental fungal ITS sequences.

BMC Bioinformatics 2017 Dec 6;18(1):538. Epub 2017 Dec 6.

Department of Plant, Soil, & Microbial Sciences, Michigan State University, East Lansing, Michigan, 48824, USA.

Background: One of the most crucial steps in high-throughput sequence-based microbiome studies is the taxonomic assignment of sequences belonging to operational taxonomic units (OTUs). Without taxonomic classification, functional and biological information of microbial communities cannot be inferred or interpreted. The internal transcribed spacer (ITS) region of the ribosomal DNA is the conventional marker region for fungal community studies. While bioinformatics pipelines that cluster reads into OTUs have received much attention in the literature, less attention has been given to the taxonomic classification of these sequences, upon which biological inference is dependent.

Results: Here we compare how three common fungal OTU taxonomic assignment tools (RDP Classifier, UTAX, and SINTAX) handle ITS fungal sequence data. The classification power, defined as the proportion of assigned OTUs at a given taxonomic rank, varied among the classifiers. Classifiers were generally consistent (assignment of the same taxonomy to a given OTU) across datasets and ranks; a small number of OTUs were assigned unique classifications across programs. We developed CONSTAX (CONSensus TAXonomy), a Python tool that compares taxonomic classifications of the three programs and merges them into an improved consensus taxonomy. This tool also produces summary classification outputs that are useful for downstream analyses.

Conclusions: Our results demonstrate that independent taxonomy assignment tools classify unique members of the fungal community, and greater classification power is realized by generating consensus taxonomy of available classifiers with CONSTAX.
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http://dx.doi.org/10.1186/s12859-017-1952-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5719527PMC
December 2017

Bifiguratus adelaidae, gen. et sp. nov., a new member of Mucoromycotina in endophytic and soil-dwelling habitats.

Mycologia 2017 May-Jun;109(3):363-378. Epub 2017 Aug 8.

a Department of Biological Sciences , Western Illinois University , Macomb , Illinois 61455.

Illumina amplicon sequencing of soil in a temperate pine forest in the southeastern United States detected an abundant, nitrogen (N)-responsive fungal genotype of unknown phylogenetic affiliation. Two isolates with ribosomal sequences consistent with that genotype were subsequently obtained. Examination of records in GenBank revealed that a genetically similar fungus had been isolated previously as an endophyte of moss in a pine forest in the southwestern United States. The three isolates were characterized using morphological, genomic, and multilocus molecular data (18S, internal transcribed spacer [ITS], and 28S rRNA sequences). Phylogenetic and maximum likelihood phylogenomic reconstructions revealed that the taxon represents a novel lineage in Mucoromycotina, only preceded by Calcarisporiella, the earliest diverging lineage in the subphylum. Sequences for the novel taxon are frequently detected in environmental sequencing studies, and it is currently part of UNITE's dynamic list of most wanted fungi. The fungus is dimorphic, grows best at room temperature, and is associated with a wide variety of bacteria. Here, a new monotypic genus, Bifiguratus, is proposed, typified by Bifiguratus adelaidae.
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http://dx.doi.org/10.1080/00275514.2017.1364958DOI Listing
May 2018