Corresponding author: Gareth W. Griffith ( gwg@aber.ac.uk ) Academic editor: Hugo de Boer
© 2020 Lina A. Clasen, Andrew P. Detheridge, John Scullion, Gareth W. Griffith.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Clasen LA, Detheridge AP, Scullion J, Griffith GW (2020) Soil stabilisation for DNA metabarcoding of plants and fungi. Implications for sampling at remote locations or via third-parties. Metabarcoding and Metagenomics 4: e58365. https://doi.org/10.3897/mbmg.4.58365
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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.
chitinolytic fungi, freeze-drying, freeze-thaw, sample preservation
The use of eDNA (environmental DNA) metabarcoding (amplicon sequencing) has transformed our knowledge of the structure and composition of soil biological communities (
As the use of eDNA metabarcoding has extended to the study of soils in more remote locations (
There are established international guidelines recommending refrigerated storage or freezing (
Current standards have been established through the International Organization for Standardization (ISO), for example
Freeze drying (with subsequent frozen storage) is widely considered by many to be the best available option for the stabilisation of soils prior to nucleic acid extraction (
Where it is not possible to freeze samples within a few hours of collection with the ability to subsequently freeze dry, the question remains as to what pre-treatment is best to preserve the nucleic acids of the soil communities during shipping from field sites (often sampled by third parties) to analytical labs. Here, we compare the effect of immediate freezing to a range of different soil DNA stabilisation methods, using equipment available outside laboratories (freezers, fridges, fans and ovens). The resulting effects of these soil storage methods are examined using eDNA metabarcoding profiles for plants and fungi, hypothesising that inferior storage conditions would lead to: variations in community structure of both plants and fungi, due to DNA degradation; proliferation of a subpopulation of faster-growing fungi, well-suited to growth in particular storage conditions, which would be associated with greater levels of DNA degradation.
Soil was collected from an upland (grazed) grassland immediately adjacent to the Brignant long-term grazing experimental field site (lat/long: 52.3648°N, 3.8214°W; 367 m a.s.l.) near Aberystwyth Wales. Brignant soil is an acidic (pH 5) Manod Series soil with loam over shale Palaeozoic slate, mudstone and siltstone (well-drained fine loamy or fine silty soils over rock; (
The soil treatments are shown in Table
Storage treatments tested in this study using soil adjacent to the Brignant long-term experiment. An “*” indicates treatments also tested with soil from the Gogerddan alluvial plain.
Treatment | Initial processing | Duration | Secondary processing | Duration | Final processing |
---|---|---|---|---|---|
T1* | Freeze -80 °C | Freeze Dry | |||
T2 | Freeze -20 °C | Overnight | Thaw.Cold room 4 °C closed bag | 14 days | Freeze -80 °C & Freeze Dry |
T3* | Freeze -20 °C | Overnight | Thaw.Cold room 4 °C closed bag | 5 days | Freeze -80 °C & Freeze Dry |
T4 | Freeze -20 °C | Overnight | Thaw.RT 23 °C closed bag | 14 days | Freeze -80 °C & Freeze Dry |
T5 | Cold room 4 °C closed bag | 3 days | RT 23 °C active air dry | 5 days | Freeze -80 °C & Freeze Dry |
T6* | Cold room 4 °C closed bag | 3 days | RT 23 °C open bag | 5 days | Freeze -80 °C & Freeze Dry |
T7 | Cold room 4 °C closed bag | 3 days | Warm dry 37 °C open bag | 5 days | Freeze -80 °C & Freeze Dry |
T8* | Cold room 4 °C closed bag | 14 days | Freeze -80 °C & Freeze Dry | ||
T9* | RT 23 °C closed bag | 14 days | Freeze -80 °C & Freeze Dry | ||
T10 | RT 23 °C closed bag | 5 days | Freeze -80 °C & Freeze Dry |
After the storage treatments were completed, all bags were frozen at -80 °C. Samples were then freeze-dried (LTE Scientific Lyotrap, 1 mbar at -50 °C for 48 h) before sieving at 0.5 mm and thoroughly homogenised, according to our standard lab procedure (
PCR was carried out using PCR Biosystems Ultra polymerase mix (PCR Biosystems Ltd, London, UK). Each 25 µl reaction contained 250 nM of the forward primer mix and 250 nM of reverse primer and 2 µl extracted soil DNA. Amplification conditions were initial denaturing 15 min at 95 °C, followed by 30 cycles at 95 °C for 30 s, 55 °C annealing for 30 s, 72 °C extension for 30 s and a final extension of 5 min at 72 °C.
After PCR, samples were quantified using the Qubit fluorometer v2 and the double stranded broad range probe (Thermo Fisher) and pooled in equal concentrations. The pooled library was cleaned using Ampure XP beads (Beckman) at a concentration of 0.65× to remove < 250 bp fragments. The libraries were then quality checked and quantified using the 2100 Bioanalyser system with a high sensitivity DNA chip (Agilent). The quantified library was diluted to 40 pM concentration and loaded on the Ion Chef and Ion PGM systems (Thermo Fisher) using the manufacturers protocol.
Sequence data were quality checked, demuliplexed and rarefied using MOTHUR (v. 1.31.2; (
Principal coordinate ordination (PCO) visualised differences in community structure using square root transformed abundances and a Bray-Curtis distance matrix; these analyses were undertaken in R (
Analysis of the functional grouping was undertaken using FUNguild (
After quality checking, there was a total of 2 772 707 ITS2 sequences with a maximum of 116 390 sequences per sample and a minimum of 44 864 (Mean 69 635). After rarefying to the lowest number of sequences per sample, dropping singleton sequences and trimming 5.8S and 28S regions, clustering resulted in 787 fungal and 60 plant OTUs for the upland (Brignant) soil and 652 fungal and 113 plant OTUs for the alluvial (Gogerddan) soil. Rarefaction curves (Suppl. material
Fungal communities detected in soils that were subject to a freeze-thaw step (T2, T3, T4) clearly diverged in PCO ordination from the fungal community in the control samples (T1). Soil samples passively air-dried at 37 °C (T7) were also strongly divergent (Fig.
Principal coordinate diagrams of the fungal community data (A) and plant community data (B) highlighting the difference in community between the different soil storage treatments (n = 4). Points show the mean axis scores and error bars show standard error of the mean.
Similar analyses for the effect of different treatments on the plant DNA (including algae: Chlorophyta) remaining after storage show that the general level of divergence from the control was less than for fungi, but still significant (Permanova Pseudo F = 2.5117 P = 0.001). Here too, freeze-thaw treatments were also the most divergent (Figs
Levels of t-statistic from pairwise Permanova of the control treatment compared to all other treatments (n = 4). The p value is shown above the bar with significant (P < 0.05) values shown in red. (A) fungal community data (B) plant community data.
Apart from the treatments mentioned above, most treatments involving storage of soil at 4 °C or at ambient temperature for up to 14 d did not result in significant changes to the plant or fungal populations later detected. In PCO ordination, the 4 °C for 14 d (T8) treatment was closest to the control for both plants and fungi (Fig.
Some storage treatments led to a reduction in fungal species diversity (Suppl. material
More detailed examination of the differences in the fungal community composition with treatment reveal a large increase in abundance of Ascomycota relative to Basidiomycota for treatment 4 (Mean 2.83) compared to the control treatment (Mean 0.70) with all other treatments remaining very similar to the control (Fig.
Variations in relative abundance of key fungal groups by storage treatment A) Ratio of Ascomycota to Basidiomycota; B) Metarhizium carneum; C) Mortierellomycotina; D) Saprotrophic fungi; E) Glomeromycotina (Arbuscular mycorrhizal fungi); F) Grassland fungi (CHEGD) (n = 4). Letters above the bars indicate significant groupings as determined by Tukey’s HSD post hoc test and error bars show standard error of the mean.
Analysis of the functional grouping, as determined unambiguously by FUNguild (
As might be expected following disruption of active plant hosts, abundance of arbuscular mycorrhizal fungi (AMF; subphylum Glomeromycotina) was reduced under the three freeze-thaw storage conditions, with a 6-fold reduction in T4. Other fungi suspected to be mycorrhizal or with intricate biotrophic association with higher plants also showed large reductions in abundance, notably the CHEGD grassland macrofungi. Combined abundance of CHEGD fungi was 5-fold lower in treatment T4 and also significantly lower in treatments T2, T3 and T7 (Fig.
Apart from green algae (Chlorophyta) which comprised < 1% of the total plant DNA in most treatments, the plant DNA present in the sieved soils was mainly within dead or dying tissues (e.g. fine roots). In contrast, a significant component of the fungal community would likely remain viable in the short term, with some species proliferating if storage conditions are conducive to their growth. Since fungi are the main decomposers of plant-derived lignocellulose in soil in terrestrial ecosystems, it is likely that proliferation of certain fungi would be associated with more rapid degradation of plant DNA. The relative sequence abundance of fungi and plants did significantly vary between treatments (Fig.
Relative sequence abundance of fungi to plants by treatment (n = 4). Letters on the bars indicate significant groupings as determined by Tukey’s HSD post hoc test and error bars show standard error of the mean.
Grasses (Poaceae; 9 spp.) were dominant (mean 88.8% of plant sequences in control treatment T1), followed by Brassicaceae (Cardamine pratensis; 5.57%), Asteraceae (3 spp.; 2.58%) and Trifolium repens (Fabaceae; 1.33%), with algae (Chlorophyta) comprising 0.41% of the plant sequences in the control soil. The turf layer was removed during sample collection, so the higher plant tissues comprised mainly (live or dead) root tissues. Several species (e.g. Crepis capillaris, Hypochaeris radicata, Ranunculus repens, Cerastium glomeratum) were detected in three or fewer of the initial 40 sieved soil samples, probably due to heterogeneous distribution of larger pieces of taproot tissue. The abundance of Poaceae varied by treatment (Suppl. material
A subset of the storage treatments (T1, T3, T6, T8 and T9) were applied to a contrasting soil type from an arable field in Gogerddan. The organic matter content of this soil was much lower (3.6% vs. 7.3%) and initial plant and fungal populations of the original soils were very different. For example, Ascomycota fungi comprised ca. 70% of the initial fungal population at Gogerddan (vs. 37% at Brignant), mostly due to the much lower abundance of CHEGD fungi (16% vs. 41%, with Hygrophoraceae absent).
As with the Brignant soils, freeze-thaw storage (T3; freeze-thaw followed by 5 d at 23 °C) resulted in the greatest difference in fungal populations relative to the control (Fig.
Principal coordinate diagrams of the fungal community data (A) and plant community data (B) in Gogerddan soil, highlighting the difference in community between the different soil storage treatments (n = 4). Points show the mean axis scores and error bars indicate standard error of the mean. The control (immediate freezing at -80 ᴼC) is indicated.
In contrast to the Brignant soil, the relative abundance of Ascomycota:Basidiomycota did not increase following freeze-thaw storage (Suppl. material
The plant community was also less diverse in the alluvial soil, with Ranunculus bulbosus (36.9%), the chlorophyte Coelastrella sp. (22.4%), Holcus lanatus (7.4%) and Polygonum aviculare (5.7%) found as the dominant plant species. Plant sequences comprised 14%–42% of all the sequences retrieved, suggesting a similar ratio of plant to fungal biomass to that found in the Brignant soil, although with a greater abundance of Chlorophyta. The dominant chlorophyte, Coelastrella sp, is a ubiquitous species found in many substrates, including soils, worldwide (
In this investigation, we have tested the effectiveness of different soil storage conditions in stabilising fungal and plant DNA prior to later storage (-80 °C) and DNA extraction. This is a concern for soil ecologists, since transport from remote and field sites to research laboratories requires interim storage in transit. This may also be a concern where soil sampling is undertaken by third parties and requires transport by mail or courier. For example, the authors recently studied the soils of endemic woodlands in St. Helena and transport of samples to Wales involved storage of the samples for up to eight days at 4 °C in sealed plastic bags (
The data presented here shows clearly that refrigerated storage for up to 14 days prior to frozen storage at -80 °C has little effect on the fungal or plant DNA later extracted. In contrast, samples initially frozen, but allowed to thaw, show the most rapid deterioration, presumably due to initial ice-damage from freezing and subsequent enzymatic degradation of DNA.
Air-drying (sometimes with the aid of silica gel) is widely used in botanical fieldwork for preservation of plant tissues (
A few fungi were observed to increase in abundance under some storage treatments, presumably because the storage conditions were conducive to their growth. Metarhizium (formerly Paecilomyces) carneum (
Of the 15 Mortierella spp. detected within the Brignant soil, all but one increased in abundance in the freeze-thawed soil incubated at 4 °C (Fig.
Of the taxa which declined substantially following freeze-thawing, Glomeromycotina (AMF) showed the greatest decline. However, within this subphylum, some taxa were more heavily affected than others. For instance, Acaulospora sp. showed > 4-fold decline (treatments T2 and T4), whereas Claroideoglomus spp. declined less than 2-fold. This is consistent with the findings of
The CHEGD fungi (barring Entolomataceae) also showed substantial decline in relative abundance in freeze-thaw treatments. Like AMF, these fungi are obligate root-associated biotrophs (
To our knowledge, this is the only study to have examined the effects of sub-optimal soil storage on eukaryotic eDNA using a high resolution method. When analysing fungal communities, for those situations where freezing samples and freeze drying are impractical, such as remote locations without equipment and requiring lengthy shipping times, the analysis indicates that the best options available are to ship cold or, if impractical, to air dry at room temperature prior to shipping. Air drying can be enhanced by using an unheated active air source, such as a blower or a fan. Though not tested here, it is likely that conditions, suitable for preservation of fungal populations, may also be appropriate for prokaryote communities, as found by
This study was supported by a grant to GWG, APD, LAC and JS from the Welsh Government (Contract C343/2017/2108; “Higher Plant DNA Sequencing in Soil” via David Rogerson and Geraint Lewis) and capacity developed as part of the Welsh European Funding Office Flexis West project C80835 (APD and JS). The Institute of Biological, Environmental and Rural Sciences receives strategic funding from the BBSRC.