CONSTAX2: improved taxonomic classification of environmental DNA markers.

SUMMARY: CONSTAX-the CONSensus TAXonomy classifier-was developed for accurate and reproducible taxonomic annotation of fungal rDNA amplicon sequences and is based upon a consensus approach of RDP, SINTAX and UTAX algorithms. CONSTAX2 extends these features to classify prokaryotes as well as eukaryotes and incorporates BLAST-based classifiers to reduce classification errors. Additionally, CONSTAX2 implements a conda-installable command-line tool with improved classification metrics, faster training, multithreading support, capacity to incorporate external taxonomic databases and new isolate matching and high-level taxonomy tools, replete with documentation and example tutorials. AVAILABILITY AND IMPLEMENTATION: CONSTAX2 is available at https://github.com/liberjul/CONSTAXv2, and is packaged for Linux and MacOS from Bioconda with use under the MIT License. A tutorial and documentation are available at https://constax.readthedocs.io/en/latest/. Data and scripts associated with the manuscript are available at https://github.com/liberjul/CONSTAXv2_ms_code. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Phyllosphere Microbiome.

The aboveground parts of terrestrial plants are colonized by a variety of microbes that collectively constitute the phyllosphere microbiota. Decades of pioneering work using individual phyllosphere microbes, including commensals and pathogens, have provided foundational knowledge about how individual microbes adapt to the phyllosphere environment and their role in providing biological control against pathogens. Recent studies have revealed a more complete repertoire of phyllosphere microbiota across plant taxa and how plants respond to and regulate the level and composition of phyllosphere microbiota. Importantly, the development of several gnotobiotic systems is allowing causative and mechanistic studies to determine the contributions of microbiota to phyllosphere health and productivity. New insights into how the phyllosphere carries out key biological processes, including photosynthesis, biomass accumulation, reproduction, and defense against biotic and abiotic insults, in either the presence or absence of a normal microbiota could unleash novel plant- and microbiota-based technologies to improve agriculturally relevant traits of crop plants.

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.

Maple and hickory leaf litter fungal communities reflect pre-senescent leaf communities.

Fungal communities are known to contribute to the functioning of living plant microbiomes as well as to the decay of dead plant material and affect vital ecosystem services, such as pathogen resistance and nutrient cycling. Yet, factors that drive structure and function of phyllosphere mycobiomes and their fate in leaf litter are often ignored. We sought to determine the factors contributing to the composition of communities in temperate forest substrates, with culture-independent amplicon sequencing of fungal communities of pre-senescent leaf surfaces, internal tissues, leaf litter, underlying humus soil of co-occurring red maple (Acer rubrum) and shagbark hickory (Carya ovata). Paired samples were taken at five sites within a temperate forest in southern Michigan, USA. Fungal communities were differentiable based on substrate, host species, and site, as well as all two-way and three-way interactions of these variables. PERMANOVA analyses and co-occurrence of taxa indicate that soil communities are unique from both phyllosphere and leaf litter communities. Correspondence of endophyte, epiphyte, and litter communities suggests dispersal plays an important role in structuring fungal communities. Future work will be needed to assess how this dispersal changes microbial community functioning in these niches.

Two new endophytic Atractiellomycetes, Atractidochium hillariae and Proceropycnis hameedii.

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.

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

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