Fungal endophytes and origins of decay in beech (Fagus sylvatica) sapwood

Sapwood comprises much above-ground forest biomass, but its mycobiome in living trees is largely unknown. Here, we characterize the endophytic fungal communities of the functional sapwood of young and mature living beech trees (Fagus sylvatica) at multiple scales, from within individual trees to woodland sites across the southern United Kingdom. Fungal community composition was determined using both culture-based and molecular approaches across two loci. Wood decay fungi, including those that cause heart rot, were detected in approximately 80% of all samples. Fungal community composition differed according to the survey approach (high throughput sequencing vs. isolation of fungi into culture) and between geographic location and individual trees, but no significant patterns were detected at different heights in individual trees or around their circumferences. ITS and LSU sequencing detected more distinct taxa than culturing. However, LSU primers yielded more OTUs than did ITS primers, though both identified unique OTUs. This highlights the importance of multiple survey approaches, including multiple primer pairs, for better characterisation of communities and confidence in results of endophyte studies

Identifying the “Mushroom of Immortality”: Assessing the Ganoderma Species Composition in Commercial Reishi Products

Species of Ganoderma, commonly called reishi (in Japan) or lingzhi (in China), have been used in traditional medicine for thousands of years, and their use has gained interest from pharmaceutical industries in recent years. Globally, the taxonomy of Ganoderma species is chaotic, and the taxon name Ganoderma lucidum has been used for most laccate (shiny) Ganoderma species. However, it is now known that G. lucidum sensu stricto has a limited native distribution in Europe and some parts of China. It is likely that differences in the quality and quantity of medicinally relevant chemicals occur among Ganoderma species. To determine what species are being sold in commercially available products, twenty manufactured products (e.g., pills, tablets, teas, etc.) and seventeen grow your own (GYO) kits labeled as containing G. lucidum were analyzed. DNA was extracted, and the internal transcribed spacer (ITS) region and translation elongation factor 1-alpha (tef1α) were sequenced with specific fungal primers. The majority (93%) of the manufactured reishi products and almost half of the GYO kits were identified as Ganoderma lingzhi. G. lingzhi is native to Asia and is the most widely cultivated and studied taxon for medicinal use. Illumina MiSeq sequencing of the ITS1 region was performed to determine if multiple Ganoderma species were present. None of the manufactured products tested contained G. lucidum sensu stricto, and it was detected in only one GYO kit. G. lingzhi was detected in most products, but other Ganoderma species were also present, including G. applanatum, G. australe, G. gibbosum, G. sessile, and G. sinense. Our results indicate that the content of these products vary and that better labeling is needed to inform consumers before these products are ingested or marketed as medicine. Of the 17 GYO kits tested, 11 kits contained Ganoderma taxa that are not native to the United States. If fruiting bodies of exotic Ganoderma taxa are cultivated, these GYO kits will likely end up in the environment. The effects of these exotic species to natural ecosystems needs investigation

Non-biological synthetic spike-in controls and the AMPtk software pipeline improve mycobiome data

High-throughput amplicon sequencing (HTAS) of conserved DNA regions is a powerful technique to characterize microbial communities. Recently, spike-in mock communities have been used to measure accuracy of sequencing platforms and data analysis pipelines. To assess the ability of sequencing platforms and data processing pipelines using fungal internal transcribed spacer (ITS) amplicons, we created two ITS spike-in control mock communities composed of cloned DNA in plasmids: a biological mock community, consisting of ITS sequences from fungal taxa, and a synthetic mock community (SynMock), consisting of non-biological ITS-like sequences. Using these spike-in controls we show that: (1) a non-biological synthetic control (e.g., SynMock) is the best solution for parameterizing bioinformatics pipelines, (2) pre-clustering steps for variable length amplicons are critically important, (3) a major source of bias is attributed to the initial polymerase chain reaction (PCR) and thus HTAS read abundances are typically not representative of starting values. We developed AMPtk, a versatile software solution equipped to deal with variable length amplicons and quality filter HTAS data based on spike-in controls. While we describe herein a non-biological SynMock community for ITS sequences, the concept and AMPtk software can be widely applied to any HTAS dataset to improve data quality

Understudied, underrepresented, and unknown: Methodological biases that limit detection of early diverging fungi from environmental samples.

Metabarcoding is an important tool for understanding fungal communities. The internal transcribed spacer (ITS) rDNA is the accepted fungal barcode but has known problems. The large subunit (LSU) rDNA has also been used to investigate fungal communities but available LSU metabarcoding primers were mostly designed to target Dikarya (Ascomycota + Basidiomycota) with little attention to early diverging fungi (EDF). However, evidence from multiple studies suggests that EDF comprise a large portion of unknown diversity in community sampling. Here, we investigate how DNA marker choice and methodological biases impact recovery of EDF from environmental samples. We focused on one EDF lineage, Zoopagomycota, as an example. We evaluated three primer sets (ITS1F/ITS2, LROR/LR3, and LR3 paired with new primer LR22F) to amplify and sequence a Zoopagomycota mock community and a set of 146 environmental samples with Illumina MiSeq. We compared two taxonomy assignment methods and created an LSU reference database compatible with AMPtk software. The two taxonomy assignment methods recovered strikingly different communities of fungi and EDF. Target fragment length variation exacerbated PCR amplification biases and influenced downstream taxonomic assignments, but this effect was greater for EDF than Dikarya. To improve identification of LSU amplicons we performed phylogenetic reconstruction and illustrate the advantages of this critical tool for investigating identified and unidentified sequences. Our results suggest much of the EDF community may be missed or misidentified with "standard" metabarcoding approaches and modified techniques are needed to understand the role of these taxa in a broader ecological context

Data from: A selective fungal transport organ (mycangium) maintains coarse phylogenetic congruence between fungus-farming ambrosia beetles and their symbionts

Thousands of species of ambrosia beetles excavate tunnels in wood to farm fungi. They maintain associations with particular lineages of fungi, but the phylogenetic extent and mechanisms of fidelity are unknown. We test the hypothesis that selectivity of their mycangium enforces fidelity at coarse phylogenetic scales, while permitting promiscuity among closely related fungal mutualists. We confirm a single evolutionary origin of the Xylosandrus complex – a group of several xyleborine genera that farm fungi in the genus Ambrosiella. Multi-level co-phylogenetic analysis revealed frequent symbiont switching within major Ambrosiella clades, but not between clades. The loss of the mycangium in Diuncus, a genus of evolutionary cheaters, was commensurate with loss of fidelity to fungal clades, supporting the hypothesis that the mycangium reinforces fidelity. Finally, in-vivo experiments tracked symbiotic compatibility throughout the symbiotic life cycle of Xylosandrus compactus and demonstrated that closely related Ambrosiella symbionts are interchangeable, but the probability of fungal uptake in the mycangium was significantly lower in more phylogenetically distant species of symbionts. Symbiont loads in experimental subjects were similar to wild-caught beetles. We conclude that partner choice in ambrosia beetles is achieved biochemically in the mycangium, and co-phylogenetic inferences can be used to predict the likelihood of specific symbiont switches

Anthelmintic drugs modulate the acute phase immune response but not the microbiota in wild Song Sparrows

Co-infection with microparasites (e.g., bacteria) and macroparasites (e.g., helminths) is often the natural state for wild animals. Despite evidence that gut helminths can bias immune responses away from inflammatory processes, few field studies have examined the role that helminths, or their potential interactions with internal microbial communities, play in modulating immunity in free-living, wild birds. Here, we used anthelmintic drugs to treat wild Song Sparrows (Melospiza melodia) for helminth infections and measured markers of systemic inflammation (heterothermia and locomotor activity) in response to an immune challenge with lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria. Using birds from a population that previously showed high helminth prevalence, we monitored skin temperature and activity remotely using automated radio telemetry. We also collected cloacal swabs to determine whether drug treatment was associated with changes in the cloacal microbiota, and whether cloacal microbial community structure was associated with the severity of birds\u27 immune responses. Because helminths can reduce the severity of inflammatory immune responses in other species, we predicted that in comparison with untreated control birds, anthelmintic-treated birds would be more lethargic and display higher fevers when challenged with LPS. Consistent with these predictions, anthelmintic-treated birds expressed higher fevers in response to immune challenge. However, all LPS-challenged birds decreased locomotor activity to a similar degree, regardless of anthelmintic treatment. Although several individual indicator bacterial taxa were strongly associated with anthelmintic treatment, this treatment did not alter overall bacterial alpha- and beta- diversity. Similarly, we did not find evidence that bacterial community diversity influenced the severity of immune responses to LPS. These results suggest that under field conditions, natural helminth infection can reduce the severity of songbirds\u27 thermoregulatory responses (fever) during an immune challenge, without major impacts on internal microbial communities or behavioral responses to infection

A global review of the ecological significance of symbiotic associations between birds and fungi

Symbiotic associations between mammals and fungi have been well documented and are widely regarded as vital to ecosystem functions around the world. Symbioses between birds and fungi are also ecologically vital but have been far less thoroughly studied. This manuscript is the first to review a wide range of symbiotic associations between birds and fungi. We compile the largest list to date of bird species reported to eat fungi (54 bird species in 27 families) and follow up with a discussion of these symbioses and suggestions for how future studies can determine the prevalence of associations between birds and fungi. We review the importance of fungi for cavity-excavating birds and show that at least 30 bird species in three families form varying levels of associations with fungi for cavity excavation. We also review the use of fungal rhizomorphs in nest construction and show that 176 bird species in 37 families use fungal material in their nests. All of these interactions have wide-reaching ecosystem implications, particularly in regard to fungal dispersal and biogeography, plant health, ecosystem function, bird nutrition/fitness and bird behaviour

Amplicon-Based Sequencing of Soil Fungi from Wood Preservative Test Sites

Soil samples were collected from field sites in two AWPA (American Wood Protection Association) wood decay hazard zones in North America. Two field plots at each site were exposed to differing preservative chemistries via in-ground installations of treated wood stakes for approximately 50 years. The purpose of this study is to characterize soil fungal species and to determine if long term exposure to various wood preservatives impacts soil fungal community composition. Soil fungal communities were compared using amplicon-based DNA sequencing of the internal transcribed spacer 1 (ITS1) region of the rDNA array. Data show that soil fungal community composition differs significantly between the two sites and that long-term exposure to different preservative chemistries is correlated with different species composition of soil fungi. However, chemical analyses using ICP-OES found levels of select residual preservative actives (copper, chromium and arsenic) to be similar to naturally occurring levels in unexposed areas. A list of indicator species was compiled for each treatment-site combination; functional guild analyses indicate that long-term exposure to wood preservatives may have both detrimental and stimulatory effects on soil fungal species composition. Fungi with demonstrated capacity to degrade industrial pollutants were found to be highly correlated with areas that experienced long-term exposure to preservative testing

Wood-colonizing fungal community response to forest restoration thinnings in a Pinus tabuliformis plantation in northern China

Forest restoration thinning in Chinese pine (Pinus tabuliformis) plantations can alter stand structure and soil abiotic properties, which have the potential to change biotic properties such as wood-inhabiting fungal community structure. Therefore, three thinning treatments (30%, 41% and 53% of the standing biomass removed) and an unthinned control stand were established at stand age 35 to determine the effects on surface and mineral soil wood-inhabiting fungi. Chinese pine, loblolly pine (Pinus taeda), and trembling aspen (Populus tremuloides) wood stakes were placed horizontally on the soil surface and inserted vertically into the mineral soil and sampled over 3 years. Fungal species were identified using high-throughput amplicon sequencing (HTAS) of DNA barcode regions. Across all wood stake species on the soil surface, operational taxonomic unit (OTU) richness averaged 138 OTUs per thinning treatment. Significantly greater OTU richness (p \u3c 0.01) on both surface pine species stakes was observed in the unthinned control as compared to the moderate and heavily thinned plots. In the mineral soil, wood stakes averaged only 91 OTUs, with no clear OTU pattern for thinning treatment or sampling time for richness. However, aspen stakes had significantly lower (p \u3c 0.01) OTU richness than both pine species stakes on the surface and in mineral soil. Although richness was not strongly affected, fungal community composition in the mineral soil was significantly altered by thinning treatments, wood stake species, and sampling time. This study extends our knowledge of the long-term effects of stand thinning on fungal communities’ richness and composition

Fungicide-Mediated Shifts in the Foliar Fungal Community of an Invasive Grass

Invasive plants, which cause substantial economic and ecological impacts, acquire both pathogens and beneficial microbes in their introduced ranges. Communities of fungal endophytes are known to mediate impacts of pathogens on plant fitness but few studies have examined the temporal dynamics of fungal communities on invasive plants. The annual grass Microstegium vimineum, an invader of forests and riparian areas throughout the eastern United States, experiences annual epidemics of disease caused by Bipolaris pathogens. Our objective was to characterize the dynamics of foliar fungal communities on M. vimineum over a growing season during a foliar disease epidemic. First, we asked how the fungal community in the phyllosphere changed over 2 months that corresponded with increasing disease severity. Second, we experimentally suppressed disease with fungicide in half of the plots and asked how the treatment affected fungal community diversity and composition. We found increasingly diverse foliar fungal communities and substantial changes in community composition between timepoints using high-throughput amplicon sequencing of the internal transcribed spacer 2 region. Monthly fungicide application caused shifts in fungal community composition relative to control samples. Fungicide application increased diversity at the late-season timepoint, suggesting that it suppressed dominant fungicide sensitive taxa and allowed other fungal taxa to flourish. These results raise new questions regarding the roles of putative endophytes found in the phyllosphere given the limited number of pathogens known to cause disease on M. vimineum in its invasive range