Soil stabilisation for DNA metabarcoding of plants and fungi. Implications for sampling at remote locations or via third-parties

Storage of soil samples prior to metagenomic analysis presents a problem. If field sites are remote or if samples are collected by third parties, transport to analytical laboratories may take several days or even weeks. The bulk of such samples and requirement for later homogenisation precludes the convenient use of a stabilisation buffer, so samples are usually cooled or frozen during transit. There has been limited testing of the most appropriate storage methods for later study of soil organisms by eDNA approaches. Here we tested a range of storage methods on two contrasting soils, comparing these methods to the control of freezing at -80 °C, followed by freeze-drying. To our knowledge, this is the first study to examine the effect of storage conditions on eukaryote DNA in soil, including both viable organisms (fungi) and DNA contained within dying/dead tissues (plants). For fungi, the best storage regimes (closest to the control) were storage at 4 °C (for up to 14 d) or active air-drying at room temperature. The worst treatments involved initial freezing, followed by thawing which led to significant later spoilage. The key spoilage organisms were identified as Metarhizium carneum and Mortierella spp., with a general increase in saprotrophic fungi and reduced abundances of mycorrhizal/biotrophic fungi. Plant data showed a similar pattern, but with greater variability in community structure, especially in the freeze-thaw treatments, probably due to stochastic variation in substrates for fungal decomposition, algal proliferation and some seed germination. In the absence of freeze drying facilities, samples should be shipped refrigerated, but not frozen if there is any risk of thawing

The legacy effect of cover crops on soil fungal populations in a cereal rotation

AbstractThe use of rotations and minimum tillage in agriculture can permit more sustainable production through increasing soil organic matter and nutrients, and breaking of pathogen lifecycles. Soil fungal populations make an important physical and chemical contribution to soil. For example, mycorrhizal species are important in plant nutrition but are often overlooked when considering management practices for efficient soil function. We undertook DNA metabarcoding (Ion Torrent) using novel PCR primers and high-throughput sequencing of the D1 region of the large ribosomal subunit of the rRNA locus, to assess the effect of different forages and cereal tillage methods on the soil fungal community. The study comprised five forage treatments, perennial ryegrass (Lolium perenne) with either low or high N, chicory (Cichorium intybus), red clover (Trifolium pratense) or white clover (Trifolium repens) grown over 3 harvest years (2010–2012). Cultivation of chicory, red clover or white clover led to significantly divergent soil fungal communities, with a notably lower diversity of fungal populations under clover, suggesting a link to soil N dynamics. Consistent with this, was a negative correlation of soil nitrate-N levels with populations of arbuscular mycorrhizal fungi (AMF) and other root-associated fungal groupings (dark septate endophytes, ‘CHEG’, Sebacinales and Ceratobasidiaceae). In contrast, abundance of Fungi belonging to the genera Mortierella and Cryptococcus were positively correlated with soil nitrate-N, with Mortierella also being negatively correlated with soil P. Spring wheat was sown on the same plots (April 2013) followed by winter barley (October 2013). Half of each plot was sown either after ploughing or by direct drilling. A legacy effect of the preceding forage crop on the fungal community was detected after both cereal crops, with plots previously cultivated with ryegrass being most divergent. No overall effect of establishment method on fungal communities was detected but AMF and CHEG fungi were more abundant on direct-drilled plots and pathogenic fungi were more abundant on ploughed plots after the sowing of winter barley

Taxon interactions control the distributions of cryoconite bacteria colonizing a High Arctic ice cap

Microbial colonization of glacial ice surfaces incurs feedbacks which affect the melting rate of the ice surface. Ecosystems formed as microbe-mineral aggregates termed cryoconite locally reduce ice surface albedo and represent foci of biodiversity and biogeochemical cycling. Consequently, greater understanding the ecological processes in the formation of functional cryoconite ecosystems upon glacier surfaces is sought. Here we present the first bacterial biogeography of an ice cap, evaluating the respective roles of dispersal, environmental and biotic filtration occurring at local scales in the assembly of cryoconite microbiota. 16S rRNA gene amplicon semiconductor sequencing of cryoconite colonizing a Svalbard ice cap coupled with digital elevation modelling of physical parameters reveals the bacterial community is dominated by a ubiquitous core of generalist taxa, with evidence for a moderate pairwise distance-decay relationship. While geographic position and melt season duration are prominent among environmental predictors of community structure, the core population of taxa appears highly influential in structuring the bacterial community. Taxon co-occurrence network analysis reveals a highly modular community structured by positive interactions with bottleneck taxa, predominantly Actinobacteria affiliated to isolates from soil humus. In contrast, the filamentous cyanobacterial taxon (assigned to Leptolyngbya) which dominates the community and bind together granular cryoconite are poorly connected to other taxa. While our study targeted one ice cap, the prominent role of generalist core taxa with close environmental relatives across the global cryosphere indicate discrete roles for cosmopolitan Actinobacteria and Cyanobacteria as respective keystone taxa and ecosystem engineers of cryoconite ecosystems colonizing ice caps. This article is protected by copyright. All rights reserved

Vegetation and edaphic factors influence rapid establishment of distinct fungal communities on former coal-spoil sites

We investigated re-establishment of fungal communities on eight former colliery sites in South Wales following revegetation 22?27?y earlier. Regraded bare coal-spoil was seeded to sheep-grazed grasslands, with saplings planted into coal-spoil for woodlands. Metabarcoding (28S rRNA, D1 region) of soil fungal populations showed that woodland and grassland habitats were clearly divergent but edaphic variables only weakly affected fungal community structure. Root-associated basidiomycetes dominated all habitats, with ectomycorrhizal fungi more abundant in woodlands and Clavariaceae/Hygrophoraceae (?CHEG? fungi) in grasslands. The composition of coal-spoil grassland communities resembled that of a typical upland grassland site, suggesting that propagule immigration was not a limiting factor. However, fungal biomass (ergosterol) was 3-fold lower, reflecting high bulk density and poor structure. Re-establishment of fungal communities in coal-spoil soils represents an important barometer of restoration success. From a fungal conservation perspective, such sites represent important refugia for waxcap fungi subject to habitat loss from agricultural intensificationpublishersversionPeer reviewe