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Auto Tag: Fungal communities

September 22, 2019  |  

PCR and omics based techniques to study the diversity, ecology and biology of anaerobic fungi: Insights, challenges andopportunities.

Anaerobic fungi (phylum Neocallimastigomycota) are common inhabitants of the digestive tract of mammalian herbivores, and in the rumen, can account for up to 20% of the microbial biomass. Anaerobic fungi play a primary role in the degradation of lignocellulosic plant material. They also have a syntrophic interaction with methanogenic archaea, which increases their fiber degradation activity. To date, nine anaerobic fungal genera have been described, with further novel taxonomic groupings known to exist based on culture-independent molecular surveys. However, the true extent of their diversity may be even more extensively underestimated as anaerobic fungi continue being discovered in yet unexplored gut and non-gut environments. Additionally many studies are now known to have used primers that provide incomplete coverage of the Neocallimastigomycota. For ecological studies the internal transcribed spacer 1 region (ITS1) has been the taxonomic marker of choice, but due to various limitations the large subunit rRNA (LSU) is now being increasingly used. How the continued expansion of our knowledge regarding anaerobic fungal diversity will impact on our understanding of their biology and ecological role remains unclear; particularly as it is becoming apparent that anaerobic fungi display niche differentiation. As a consequence, there is a need to move beyond the broad generalization of anaerobic fungi as fiber-degraders, and explore the fundamental differences that underpin their ability to exist in distinct ecological niches. Application of genomics, transcriptomics, proteomics and metabolomics to their study in pure/mixed cultures and environmental samples will be invaluable in this process. To date the genomes and transcriptomes of several characterized anaerobic fungal isolates have been successfully generated. In contrast, the application of proteomics and metabolomics to anaerobic fungal analysis is still in its infancy. A central problem for all analyses, however, is the limited functional annotation of anaerobic fungal sequence data. There is therefore an urgent need to expand information held within publicly available reference databases. Once this challenge is overcome, along with improved sample collection and extraction, the application of these techniques will be key in furthering our understanding of the ecological role and impact of anaerobic fungi in the wide range of environments they inhabit.

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September 22, 2019  |  

Great differences in performance and outcome of high-throughput sequencing data analysis platforms for fungal metabarcoding.

Along with recent developments in high-throughput sequencing (HTS) technologies and thus fast accumulation of HTS data, there has been a growing need and interest for developing tools for HTS data processing and communication. In particular, a number of bioinformatics tools have been designed for analysing metabarcoding data, each with specific features, assumptions and outputs. To evaluate the potential effect of the application of different bioinformatics workflow on the results, we compared the performance of different analysis platforms on two contrasting high-throughput sequencing data sets. Our analysis revealed that the computation time, quality of error filtering and hence output of specific bioinformatics process largely depends on the platform used. Our results show that none of the bioinformatics workflows appears to perfectly filter out the accumulated errors and generate Operational Taxonomic Units, although PipeCraft, LotuS and PIPITS perform better than QIIME2 and Galaxy for the tested fungal amplicon dataset. We conclude that the output of each platform requires manual validation of the OTUs by examining the taxonomy assignment values.

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September 22, 2019  |  

Soil microclimate changes affect soil fungal communities in a Mediterranean pine forest.

Soil microclimate is a potentially important regulator of the composition of plant-associated fungal communities in climates with significant drought periods. Here, we investigated the spatio-temporal dynamics of soil fungal communities in a Mediterranean Pinus pinaster forest in relation to soil moisture and temperature. Fungal communities in 336 soil samples collected monthly over 1 year from 28 long-term experimental plots were assessed by PacBio sequencing of ITS2 amplicons. Total fungal biomass was estimated by analysing ergosterol. Community changes were analysed in the context of functional traits. Soil fungal biomass was lowest during summer and late winter and highest during autumn, concurrent with a greater relative abundance of mycorrhizal species. Intra-annual spatio-temporal changes in community composition correlated significantly with soil moisture and temperature. Mycorrhizal fungi were less affected by summer drought than free-living fungi. In particular, mycorrhizal species of the short-distance exploration type increased in relative abundance under dry conditions, whereas species of the long-distance exploration type were more abundant under wetter conditions. Our observations demonstrate a potential for compositional and functional shifts in fungal communities in response to changing climatic conditions. Free-living fungi and mycorrhizal species with extensive mycelia may be negatively affected by increasing drought periods in Mediterranean forest ecosystems.© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

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September 22, 2019  |  

High-resolution community profiling of arbuscular mycorrhizal fungi.

Community analyses of arbuscular mycorrhizal fungi (AMF) using ribosomal small subunit (SSU) or internal transcribed spacer (ITS) DNA sequences often suffer from low resolution or coverage. We developed a novel sequencing based approach for a highly resolving and specific profiling of AMF communities. We took advantage of previously established AMF-specific PCR primers that amplify a c. 1.5-kb long fragment covering parts of SSU, ITS and parts of the large ribosomal subunit (LSU), and we sequenced the resulting amplicons with single molecule real-time (SMRT) sequencing. The method was applicable to soil and root samples, detected all major AMF families and successfully discriminated closely related AMF species, which would not be discernible using SSU sequences. In inoculation tests we could trace the introduced AMF inoculum at the molecular level. One of the introduced strains almost replaced the local strain(s), revealing that AMF inoculation can have a profound impact on the native community. The methodology presented offers researchers a powerful new tool for AMF community analysis because it unifies improved specificity and enhanced resolution, whereas the drawback of medium sequencing throughput appears of lesser importance for low-diversity groups such as AMF.© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

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September 22, 2019  |  

Below-ground organic matter accumulation along a boreal forest fertility gradient relates to guild interaction within fungal communities.

Plant-soil interactions link ecosystem fertility and organic matter accumulation below ground. Soil microorganisms play a central role as mediators of these interactions, but mechanistic understanding is still largely lacking. Correlative data from a coniferous forest ecosystem support the hypothesis that interactions between fungal guilds play a central role in regulating organic matter accumulation in relation to fertility. With increasing ecosystem fertility, the proportion of saprotrophic basidiomycetes increased in deeper organic layers, at the expense of ectomycorrhizal fungal species. Saprotrophs correlated positively with the activity of oxidative enzymes, which in turn favoured organic matter turnover and nitrogen recycling to plants. Combined, our findings are consistent with a fungus-mediated feedback loop, which results in a negative correlation between ecosystem fertility and below-ground carbon storage. These findings call for a shift in focus from plant litter traits to fungal traits in explaining organic matter dynamics and ecosystem fertility in boreal forests.© 2017 John Wiley & Sons Ltd/CNRS.

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September 22, 2019  |  

Fungal community shifts underpin declining mycelial production and turnover across a Pinus sylvestris chronosequence

Fungi play critical roles in ecosystem processes such as decomposition and nutrient cycling, but have also been highlighted as significant contributors to organic matter build-up in boreal forest soils. Ectomycorrhizal (ECM) mycelial biomass and necromass dynamics have recently been highlighted as essential for regulating build-up of soil organic matter. Understanding the extent to which shifts in mycelial community or growth trait composition cause changes in mycelial production and turnover over ecological gradients would aid a mechanistic understanding of these important processes at an ecosystem scale. Here, we test the hypotheses that shifting species and mycelial trait (exploration type) composition within the mycelial community underpin changes in biomass turnover with increasing forest age. We quantified mycelial turnover and assessed fungal community composition in a chronosequence of eight, 12- to 158-year-old, managed Pinus sylvestris forests. Turnover was estimated by determining mycelial biomass (ergosterol) in a sequence of ingrowth mesh bags and applying mathematical models. Fungal communities in the bags were identified using Pacific Biosciences sequencing of fungal ITS2 amplicons. To evaluate the accuracy of this method to represent all ECM fungi, community composition in bags was followed over time and compared with communities in soil. Mycelial communities changed with stand age, but we found no evidence that there were concurrent shifts in mycelial exploration types. Forest age and turnover were significantly correlated with ECM mycelial community composition and collectively explained 39.4% of total variation. The similarity between fungal communities in mesh bags and in soil was strongly forest age dependent, with communities in mesh bags diverging from soil communities in stands older than 60 years. However, in all stands, when bag incubation time exceeded 75 days, communities became more similar to soil communities. Synthesis. Our results support the idea that shifts in fungal community composition underpin the forest age-related decrease in mycelial turnover; however, since ingrowth mesh bags exclude some mycorrhizal species in older forests, it remains a possibility that turnover estimates were not reflecting the entire community. While we found no evidence that mycelial exploration types of fungi changed systematically with forest age, we suggest that other traits that relate to biomass turnover and necromass degradation require further study, as they may explain the extent to which ectomycorrhizal fungi regulate and contribute to soil organic matter accumulation.

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September 22, 2019  |  

Retention of seed trees fails to lifeboat ectomycorrhizal fungal diversity in harvested Scots pine forests.

Fennoscandian forestry has in the past decades changed from natural regeneration of forests towards replantation of clear-cuts, which negatively impacts ectomycorrhizal fungal (EMF) diversity. Retention of trees during harvesting enables EMF survival, and we therefore expected EMF communities to be more similar to those in old natural stands after forest regeneration using seed trees compared to full clear-cutting and replanting. We sequenced fungal internal transcribed spacer 2 (ITS2) amplicons to assess EMF communities in 10- to 60-year-old Scots pine stands regenerated either using seed trees or through replanting of clear-cuts with old natural stands as reference. We also investigated local EMF communities around retained old trees. We found that retention of seed trees failed to mitigate the impact of harvesting on EMF community composition and diversity. With increasing stand age, EMF communities became increasingly similar to those in old natural stands and permanently retained trees maintained EMF locally. From our observations, we conclude that EMF communities, at least common species, post-harvest are more influenced by environmental filtering, resulting from environmental changes induced by harvest, than by the continuity of trees. These results suggest that retention of intact forest patches is a more efficient way to conserve EMF diversity than retaining dispersed single trees.© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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September 22, 2019  |  

Soil drying procedure affects the DNA quantification of Lactarius vinosus but does not change the fungal community composition.

Drying soil samples before DNA extraction is commonly used for specific fungal DNA quantification and metabarcoding studies, but the impact of different drying procedures on both the specific fungal DNA quantity and the fungal community composition has not been analyzed. We tested three different drying procedures (freeze-drying, oven-drying, and room temperature) on 12 different soil samples to determine (a) the soil mycelium biomass of the ectomycorrhizal species Lactarius vinosus using qPCR with a specifically designed TaqMan® probe and (b) the fungal community composition and diversity using the PacBio® RS II sequencing platform. Mycelium biomass of L. vinosus was significantly greater in the freeze-dried soil samples than in samples dried at oven and room temperature. However, drying procedures had no effect on fungal community composition or on fungal diversity. In addition, there were no significant differences in the proportions of fungi according to their functional roles (moulds vs. mycorrhizal species) in response to drying procedures. Only six out of 1139 operational taxonomic units (OTUs) had increased their relative proportions after soil drying at room temperature, with five of these OTUs classified as mould or yeast species. However, the magnitude of these changes was small, with an overall increase in relative abundance of these OTUs of approximately 2 %. These results suggest that DNA degradation may occur especially after drying soil samples at room temperature, but affecting equally nearly all fungi and therefore causing no significant differences in diversity and community composition. Despite the minimal effects caused by the drying procedures at the fungal community composition, freeze-drying resulted in higher concentrations of L. vinosus DNA and prevented potential colonization from opportunistic species.

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September 22, 2019  |  

Long-read DNA metabarcoding of ribosomal RNA in the analysis of fungi from aquatic environments.

DNA metabarcoding is widely used to study prokaryotic and eukaryotic microbial diversity. Technological constraints limit most studies to marker lengths below 600 base pairs (bp). Longer sequencing reads of several thousand bp are now possible with third-generation sequencing. Increased marker lengths provide greater taxonomic resolution and allow for phylogenetic methods of classification, but longer reads may be subject to higher rates of sequencing error and chimera formation. In addition, most bioinformatics tools for DNA metabarcoding were designed for short reads and are therefore unsuitable. Here, we used Pacific Biosciences circular consensus sequencing (CCS) to DNA-metabarcode environmental samples using a ca. 4,500 bp marker that included most of the eukaryote SSU and LSU rRNA genes and the complete ITS region. We developed an analysis pipeline that reduced error rates to levels comparable to short-read platforms. Validation using a mock community indicated that our pipeline detected 98% of chimeras de novo. We recovered 947 OTUs from water and sediment samples from a natural lake, 848 of which could be classified to phylum, 397 to genus and 330 to species. By allowing for the simultaneous use of three databases (Unite, SILVA and RDP LSU), long-read DNA metabarcoding provided better taxonomic resolution than any single marker. We foresee the use of long reads enabling the cross-validation of reference sequences and the synthesis of ribosomal rRNA gene databases. The universal nature of the rRNA operon and our recovery of >100 nonfungal OTUs indicate that long-read DNA metabarcoding holds promise for studies of eukaryotic diversity more broadly.© 2018 John Wiley & Sons Ltd.

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September 22, 2019  |  

Root endophytes and invasiveness: no difference between native and non-native Phragmites in the Great Lakes Region

Microbial interactions could play an important role in plant invasions. If invasive plants associate with relatively more mutualists or fewer pathogens than their native counterparts, then microbial communities could foster plant invasiveness. Studies examining the effects of microbes on invasive plants commonly focus on a single microbial group (e.g., bacteria) or measure only plant response to microbes, not documenting the specific taxa associating with invaders. We surveyed root microbial communities associated with co-occurring native and non-native lineages of Phragmites australis, across Michigan, USA. Our aim was to determine whether (1) plant lineage was a stronger predictor of root microbial community composition than environmental variables and (2) the non-native lineage associated with more mutualistic and/or fewer pathogenic microbes than the native lineage. We used microscopy and culture-independent molecular methods to examine fungal colonization rate and community composition in three major microbial groups (bacteria, fungi, and oomycetes) within roots. We also used microbial functional databases to assess putative functions of the observed microbial taxa. While fungal colonization of roots was significantly higher in non-native Phragmites than the native lineage, we found no differences in root microbial community composition or potential function between the two Phragmites lineages. Community composition did differ significantly by site, with soil saturation playing a significant role in structuring communities in all three microbial groups. The relative abundance of some specific bacterial taxa did differ between Phragmites lineages at the phylum and genus level (e.g., Proteobacteria, Firmicutes). Purported function of root fungi and respiratory mode of root bacteria also did not differ between native and non-native Phragmites. We found no evidence that native and non-native Phragmites harbored distinct root microbial communities; nor did those communities differ functionally. Therefore, if the trends revealed at our sites are widespread, it is unlikely that total root microbial communities are driving invasion by non-native Phragmites plants.

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September 22, 2019  |  

Soil bacterial communities are shaped by temporal and environmental filtering: evidence from a long-term chronosequence.

Soil microbial communities are abundant, hyper-diverse and mediate global biogeochemical cycles, but we do not yet understand the processes mediating their assembly. Current hypothetical frameworks suggest temporal (e.g. dispersal limitation) and environmental (e.g. soil pH) filters shape microbial community composition; however, there is limited empirical evidence supporting this framework in the hyper-diverse soil environment, particularly at large spatial (i.e. regional to continental) and temporal (i.e. 100 to 1000 years) scales. Here, we present evidence from a long-term chronosequence (4000 years) that temporal and environmental filters do indeed shape soil bacterial community composition. Furthermore, nearly 20 years of environmental monitoring allowed us to control for potentially confounding environmental variation. Soil bacterial communities were phylogenetically distinct across the chronosequence. We determined that temporal and environmental factors accounted for significant portions of bacterial phylogenetic structure using distance-based linear models. Environmental factors together accounted for the majority of phylogenetic structure, namely, soil temperature (19%), pH (17%) and litter carbon:nitrogen (C:N; 17%). However, of all individual factors, time since deglaciation accounted for the greatest proportion of bacterial phylogenetic structure (20%). Taken together, our results provide empirical evidence that temporal and environmental filters act together to structure soil bacterial communities across large spatial and long-term temporal scales. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

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September 22, 2019  |  

Soil microbial communities and elk foraging intensity: implications for soil biogeochemical cycling in the sagebrush steppe.

Foraging intensity of large herbivores may exert an indirect top-down ecological force on soil microbial communities via changes in plant litter inputs. We investigated the responses of the soil microbial community to elk (Cervus elaphus) winter range occupancy across a long-term foraging exclusion experiment in the sagebrush steppe of the North American Rocky Mountains, combining phylogenetic analysis of fungi and bacteria with shotgun metagenomics and extracellular enzyme assays. Winter foraging intensity was associated with reduced bacterial richness and increasingly distinct bacterial communities. Although fungal communities did not respond linearly to foraging intensity, a greater ß-diversity response to winter foraging exclusion was observed. Furthermore, winter foraging exclusion increased soil cellulolytic and hemicellulolytic enzyme potential and higher foraging intensity reduced chitinolytic gene abundance. Thus, future changes in winter range occupancy may shape biogeochemical processes via shifts in microbial communities and subsequent changes to their physiological capacities to cycle soil C and N.© 2017 John Wiley & Sons Ltd/CNRS.

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September 22, 2019  |  

Multiplex amplicon sequencing for microbe identification in community-based culture collections.

Microbiome analysis using metagenomic sequencing has revealed a vast microbial diversity associated with plants. Identifying the molecular functions associated with microbiome-plant interaction is a significant challenge concerning the development of microbiome-derived technologies applied to agriculture. An alternative to accelerate the discovery of the microbiome benefits to plants is to construct microbial culture collections concomitant with accessing microbial community structure and abundance. However, traditional methods of isolation, cultivation, and identification of microbes are time-consuming and expensive. Here we describe a method for identification of microbes in culture collections constructed by picking colonies from primary platings that may contain single or multiple microorganisms, which we named community-based culture collections (CBC). A multiplexing 16S rRNA gene amplicon sequencing based on two-step PCR amplifications with tagged primers for plates, rows, and columns allowed the identification of the microbial composition regardless if the well contains single or multiple microorganisms. The multiplexing system enables pooling amplicons into a single tube. The sequencing performed on the PacBio platform led to recovery near-full-length 16S rRNA gene sequences allowing accurate identification of microorganism composition in each plate well. Cross-referencing with plant microbiome structure and abundance allowed the estimation of diversity and abundance representation of microorganism in the CBC.

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September 22, 2019  |  

Comparative genomics and transcriptomics depict ericoid mycorrhizal fungi as versatile saprotrophs and plant mutualists.

Some soil fungi in the Leotiomycetes form ericoid mycorrhizal (ERM) symbioses with Ericaceae. In the harsh habitats in which they occur, ERM plant survival relies on nutrient mobilization from soil organic matter (SOM) by their fungal partners. The characterization of the fungal genetic machinery underpinning both the symbiotic lifestyle and SOM degradation is needed to understand ERM symbiosis functioning and evolution, and its impact on soil carbon (C) turnover. We sequenced the genomes of the ERM fungi Meliniomyces bicolor, M. variabilis, Oidiodendron maius and Rhizoscyphus ericae, and compared their gene repertoires with those of fungi with different lifestyles (ecto- and orchid mycorrhiza, endophytes, saprotrophs, pathogens). We also identified fungal transcripts induced in symbiosis. The ERM fungal gene contents for polysaccharide-degrading enzymes, lipases, proteases and enzymes involved in secondary metabolism are closer to those of saprotrophs and pathogens than to those of ectomycorrhizal symbionts. The fungal genes most highly upregulated in symbiosis are those coding for fungal and plant cell wall-degrading enzymes (CWDEs), lipases, proteases, transporters and mycorrhiza-induced small secreted proteins (MiSSPs). The ERM fungal gene repertoire reveals a capacity for a dual saprotrophic and biotrophic lifestyle. This may reflect an incomplete transition from saprotrophy to the mycorrhizal habit, or a versatile life strategy similar to fungal endophytes.© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

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September 22, 2019  |  

Rhizospheric microbial communities are driven by Panax ginseng at different growth stages and biocontrol bacteria alleviates replanting mortality

The cultivation of Panax plants is hindered by replanting problems, which may be caused by plant-driven changes in the soil microbial community. Inoculation with microbial antagonists may efficiently alleviate replanting issues. Through high-throughput sequencing, this study revealed that bacterial diversity decreased, whereas fungal diversity increased, in the rhizosphere soils of adult ginseng plants at the root growth stage under different ages. Few microbial community, such as Luteolibacter, Cytophagaceae, Luteibacter, Sphingomonas, Sphingomonadaceae, and Zygomycota, were observed; the relative abundance of microorganisms, namely, Brevundimonas, Enterobacteriaceae, Pandoraea, Cantharellales, Dendryphion, Fusarium, and Chytridiomycota, increased in the soils of adult ginseng plants compared with those in the soils of 2-year-old seedlings. Bacillus subtilis 50-1, a microbial antagonist against the pathogenic Fusarium oxysporum, was isolated through a dual culture technique. These bacteria acted with a biocontrol efficacy of 67.8%. The ginseng death rate and Fusarium abundance decreased by 63.3% and 46.1%, respectively, after inoculation with B. subtilis 50-1. Data revealed that microecological degradation could result from ginseng-driven changes in rhizospheric microbial communities; these changes are associated with the different ages and developmental stages of ginseng plants. Biocontrol using microbial antagonists alleviated the replanting problem.

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