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

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.

Best practices in metabarcoding of fungi: From experimental design to results

“…Pooled subsamples should be well mixed before freezing, because it may subsequently be difficult to homogenize frozen material, which could lead to some parts of the sample or entire subsamples effectively being excluded from DNA extraction. When freezing, it is important to avoid thawing, which may lead to sample spoilage and significant changes in the detected communities (Anslan et al, 2021; Clasen et al, 2020). Long‐term storage (2–4 weeks) at 4°C may alter soil fungal diversity (Delavaux et al, 2020) and promote proliferation of moulds (Clasen et al, 2020).…”

“…When freezing, it is important to avoid thawing, which may lead to sample spoilage and significant changes in the detected communities (Anslan et al, 2021; Clasen et al, 2020). Long‐term storage (2–4 weeks) at 4°C may alter soil fungal diversity (Delavaux et al, 2020) and promote proliferation of moulds (Clasen et al, 2020). Rapid drying methods such as freeze drying and cabinet drying are alternatives to freezing to prevent DNA degradation (Castaño et al, 2016).…”

Isolation and Identification of Entomopathogenic Fungi from Soils of Manipur (N-E India)

“…Overall, 10 samples were collected from each district, resulting in 100 samples in total, which were subsequently transported to Tamil Nadu Agricultural University, Coimbatore for further analysis. The transportation process took several days, necessitating the preparation of the samples for transport without compromising the viability of entomopathogenic fungi, as per the methods outlined by Clasen et al, 2020. All the soil samples were stored in a refrigerator at 4-5  C until further processing. In the laboratory, each bag containing soil was thoroughly mixed and homogenized manually.…”

Choices on sampling, sequencing, and analyzing DNA influence the estimation of community composition of plant fungal symbionts

“…Although interest in fungal community ecology is typically on the abiotic and biotic filters sorting species, the specific methods used to prepare and sequence DNA may also influence species detection. For instance, sample storage (Clasen et al, 2020 ; Guerrieri et al, 2021 ), sequencing primers (Bellemain et al, 2010 ; Yang et al, 2018 ), and bioinformatic pipelines (Pauvert et al, 2019 ) can influence the community composition of soil fungi. While next‐generation sequencing methods have become almost default in current mycorrhizal research owing to their ability to sequence a mixed‐DNA template across many samples simultaneously, and with high sequencing depth (Nilsson et al, 2019 ), Sanger sequencing (i.e., sequencing DNA from individual mycorrhizal root tips) may be appropriate when the goal is detecting shifts in common fungal species (e.g., Shemesh et al, 2020 ).…”