Abstract
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.
Highlights
The use of eDNA metabarcoding has transformed our knowledge of the structure and composition of soil biological communities (Geml et al 2014; Williams 2020), with more recent metagenomic studies enhancing our understanding of the metabolic processes mediated by these organisms (Keepers et al 2019; Ogwu et al 2019)
Pairwise Permanova comparisons of the fungal populations in each treatment with the control (Fig. 2A) confirm that treatments T2, T3, T4, and T7 had a significant effect on the fungal populations present at the end of the storage period
The CHEGD fungi comprised > 40% of the total fungal biomass at the Brignant site and are recognised to be the dominant fungi of undisturbed mesotrophic grasslands (Halbwachs et al 2013; Detheridge et al 2018). Their susceptibility to freeze-thaw treatment and resultant increase in fungal necromass is likely the cause of the large proliferation of the chitinolytic M. carneum and Mortierella spp. in freeze-thaw treatments. This is the only study to have examined the effects of sub-optimal soil storage on eukaryotic eDNA using a high resolution method
Summary
The use of eDNA (environmental DNA) metabarcoding (amplicon sequencing) has transformed our knowledge of the structure and composition of soil biological communities (Geml et al 2014; Williams 2020), with more recent metagenomic studies enhancing our understanding of the metabolic processes mediated by these organisms (Keepers et al 2019; Ogwu et al 2019). As the use of eDNA metabarcoding has extended to the study of soils in more remote locations (Tedersoo et al 2014; Detheridge et al 2020) and to more applied deployment in nature conservation site monitoring by statutory organisations (Geml et al 2014; Detheridge et al 2018; Latch 2020; Valentin et al 2020), the issue of soil storage between sampling and subsequent analysis has become an important consideration For metabarcoding and metagenomic studies, the preservation of DNA or RNA unchanged from the natural state presents its own distinctive challenges
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