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

Variation in Soil Fungal Composition Associated with the Invasion of Stellera chamaejasme L. in Qinghai–Tibet Plateau Grassland

Stellera chamaejasme L. is the most problematic weed in China’s grasslands. Its root exudates affect co-occurring plants and thus may also affect soil fungi. Soils (0–20 cm depth) on two adjacent sites, one invaded the other uninvaded, were compared for a range of physiochemical parameters and by DNA sequencing of fungal communities. At the invaded site, relationships between S. chamaejasme abundance, soil physiochemical factors, and fungal communities were further investigated to determine whether these relationships corroborated conclusions on the basis of site differences that could be translated into functional variation. Results showed that the invaded soils had lower N, P, organic matter, fungal alpha diversity, and relative abundance of arbuscular mycorrhizal fungi (AMF), but greater abundance of pathogenic fungi. Organic matter and P were the edaphic factors most strongly linked to site differences in total fungal communities. Within the invaded site, organic matter rather than S. chamaejasme cover was closely linked to total fungal composition. However, on this site, a number of fungal species that had various ecological functions and that differentiated the two sites were related to S. chamaejasme cover. This study indicates that lower fertility soils may be more susceptible to invasion by S. chamaejasme. Although the influence of S. chamaejasme on total fungal community composition was limited, there was evidence of effects on particular fungal species. Further research is needed to determine whether these effects influence S. chamaejasme invasiveness

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

Variation in Soil Fungal Composition Associated with the Invasion of Stellera chamaejasme L. in Qinghai-Tibet Plateau Grassland

Stellera chamaejasme L. is the most problematic weed in China's grasslands. Its root exudates affect co-occurring plants and thus may also affect soil fungi. Soils (0-20 cm depth) on two adjacent sites, one invaded the other uninvaded, were compared for a range of physiochemical parameters and by DNA sequencing of fungal communities. At the invaded site, relationships between S. chamaejasme abundance, soil physiochemical factors, and fungal communities were further investigated to determine whether these relationships corroborated conclusions on the basis of site differences that could be translated into functional variation. Results showed that the invaded soils had lower N, P, organic matter, fungal alpha diversity, and relative abundance of arbuscular mycorrhizal fungi (AMF), but greater abundance of pathogenic fungi. Organic matter and P were the edaphic factors most strongly linked to site differences in total fungal ommunities. Within the invaded site, organic matter rather than S. chamaejasme cover was closely linked to total fungal composition. However, on this site, a number of fungal species that had various ecological functions and that differentiated the two sites were related to S. chamaejasme cover. This study indicates that lower fertility soils may be more susceptible to invasion by S. chamaejasme. Although the influence of S. chamaejasme on total fungal community composition was limited, there was evidence of effects on particular fungal species. Further research is needed to determine whether these effects influence S. chamaejasme invasiveness.This work was supported by the National Natural Science Foundation of China (31402133), Special Aid Fund for Qinghai Province (2020-QY-210), and Key Laboratory Research Fund of Department of Education of Shaanxi Province (18JS110) to W.H.; National Natural Science Foundation of China (41871335) to Y.L.; Special Fund for Agro-scientific Research in the Public Interest of China (201203062) to Y.W., W.H., and J.S. We acknowledge the support of a Stapledon Fellowship and BBSRC (Biotechnology and Biological Sciences Research Council) exchange grant BB/M027945/1, which helped in the formation of this manuscript. A.D. is supported by the WEFO/ERDF (Welsh European Funding Office/European Regional Development Fund) funded Flexis project