Fungi that produce their fruiting bodies underground within the soil profile are known commonly as truffles. Truffle fruiting bodies harbor a diverse but poorly understood microbial community of ...bacteria, yeasts, and filamentous fungi. In this study, we used next-generation 454 amplicon pyrosequencing of the V1 and V4 region of the bacterial 16S ribosomal DNA (rDNA) in order to characterize and compare effects of truffle species and geographic origin on the truffle microbiome. We compared truffle microbiomes of the glebal tissue for eight truffle species belonging to four distinct genera within the Pezizales: Tuber, Terfezia, Leucangium, and Kalapuya. The bacterial community within truffles was dominated by Proteobacteria, Bacterioides, Actinobacteria, and Firmicutes. Bacterial richness within truffles was quite low overall, with between 2–23 operational taxonomic units (OTUs). Notably, we found a single Bradyrhizobium OTU to be dominant within truffle species belonging to the genus Tuber, irrespective of geographic origin, but not in other truffle genera sampled. This study offers relevant insights into the truffle microbiome and raises questions concerning the recruitment and function of these fungal-associated bacteria consortia.
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.
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.
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.
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.
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.
Scleractinian assemblages are threatened by marine heat waves with coral survivorship depending on host genetics and microbiome composition. We documented an extreme marine heat wave in Fiji and the ...response of corals in two thermally stressed reef flats. Through high-throughput amplicon sequencing of 16S and ITS rDNA phylogenetic markers, we assessed coral microbiomes (Symbiodiniaceae, prokaryotes, fungi, and Apicomplexa) of paired bleached and unbleached colonies of four common coral species representative of dominant genera in the South Pacific. While all coral species exhibited one or more pathways to bleaching resistance, harboring assemblages composed primarily of thermally tolerant photosymbionts did not always result in host bleaching resistance. Montipora and Pocillopora species, which associate with diverse Symbiodiniaceae and vertically transmit their photosymbionts, fared better than Acropora, which acquire their photosymbionts from the environment, and Porites, which associate with a narrow photosymbiont assemblage. Prokaryotic and fungal beta diversity did not differ between bleached and unbleached conspecifics, however, the relative abundance of the fungus Malassezia globosa was significantly greater in unbleached colonies of Montipora digitata. Each coral species harbored distinct assemblages of Symbiodiniaceae, prokaryotes, and Apicomplexa, but not fungi, reiterating the importance of host genetics in structuring components of its microbiome. Terrestrial fungal and prokaryotic taxa were detected at low abundance across coral microbiomes, indicating that allochthonous microbial inputs occur, but that coral microbiomes remain dominated by marine microbial taxa. Our study offers valuable insights into the microbiome assemblages associated with coral tolerance to extreme water temperatures.
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•All four species examined hosted unique microbiome assemblages.•Host bleaching tolerance was driven by photosymbiont assemblages in some coral species, but not others.•Unbleached Montipora had a significantly greater relative abundance of the fungus Malassezia globosa.•Terrestrial fungi and prokaryotes were found in low abundance in coral microbiomes.
Species of the genus Tuber have gained a lot of attention in recent decades due to their aromatic hypogenous fruitbodies, which can bring high prices on the market. The tendency in truffle production ...is to infect oak, hazel, beech, etc. in greenhouse conditions. We aimed to show whether silver fir (Abies alba Mill.) can be an appropriate host partner for commercial mycorrhization with truffles, and how earthworms in the inoculation substrate would affect the mycorrhization dynamics. Silver fir seedlings inoculated with Tuber. aestivum were analyzed for root system parameters and mycorrhization, how earthworms affect the bare root system, and if mycorrhization parameters change when earthworms are added to the inoculation substrate. Seedlings were analyzed 6 and 12 months after spore inoculation. Mycorrhization with or without earthworms revealed contrasting effects on fine root biomass and morphology of silver fir seedlings. Only a few of the assessed fine root parameters showed statistically significant response, namely higher fine root biomass and fine root tip density in inoculated seedlings without earthworms 6 months after inoculation, lower fine root tip density when earthworms were added, the specific root tip density increased in inoculated seedlings without earthworms 12 months after inoculation, and general negative effect of earthworm on branching density. Silver fir was confirmed as a suitable host partner for commercial mycorrhization with truffles, with 6% and 35% mycorrhization 6 months after inoculation and between 36% and 55% mycorrhization 12 months after inoculation. The effect of earthworms on mycorrhization of silver fir with Tuber aestivum was positive only after 6 months of mycorrhization, while this effect disappeared and turned insignificantly negative after 12 months due to the secondary effect of grazing on ectomycorrhizal root tips.
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 ß-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.
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 alpha- and beta-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.
Abstract
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.
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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