Effects of long‐term differential fertilization on eukaryotic microbial communities in an arable soil: a multiple barcoding approach

Abstract

To understand the fine-scale effects of changes in nutrient availability on eukaryotic soil microorganisms communities, a multiple barcoding approach was used to analyse soil samples from four different treatments in a long-term fertilization experiment. We performed PCR amplification on soil DNA with primer pairs specifically targeting the 18S rRNA genes of all eukaryotes and three protist groups (Cercozoa, Chrysophyceae-Synurophyceae and Kinetoplastida) as well as the ITS gene of fungi and the 23S plastid rRNA gene of photoautotrophic microorganisms. Amplicons were pyrosequenced, and a total of 88,706 quality filtered reads were clustered into 1232 operational taxonomic units (OTU) across the six data sets. Comparisons of the taxonomic coverage achieved based on overlapping assignment of OTUs revealed that half of the eukaryotic taxa identified were missed by the universal eukaryotic barcoding marker. There were only little differences in OTU richness observed between organic- (farmyard manure), mineral- and nonfertilized soils. However, the community compositions appeared to be strongly structured by organic fertilization in all data sets other than that generated using the universal eukaryotic 18S rRNA gene primers, whereas mineral fertilization had only a minor effect. In addition, a co-occurrence based network analysis revealed complex potential interaction patterns between OTUs from different trophic levels, for example between fungivorous flagellates and fungi. Our results demonstrate that changes in pH, moisture and organic nutrients availability caused shifts in the composition of eukaryotic microbial communities at multiple trophic levels.

Parasites dominate hyperdiverse soil protist communities in Neotropical rainforests

“…For example, while the broadly targeted V4 primers used here amplify a wide variety of eukaryotic lineages 13,[25][26][27][28][29] , other primers could have amplified additional taxa [30][31][32] (Supplementary Table 1). The resolution power of metabarcoding also has its limits.…”

Tree diversity and species identity effects on soil fungi, protists and animals are context dependent

“…Our approach allows addition of further primer variants to capture additional taxonomic groups. Alternatively, different groups can be targeted in separate PCR reactions or using different markers followed by estimation of biomass or individuals by subsampling or quantitative PCR (Fierer et al, 2005;de Barba et al, 2014;Lentendu et al, 2014). Multiple pairs of MID-tagged primers and preparing several metabarcoding libraries per Effects of tree diversity and species identity on soil biota L Tedersoo et al sample dramatically enhance requirements for time and analytical costs.…”

Linking molecular deadwood-inhabiting fungal diversity and community dynamics to ecosystem functions and processes in Central European forests

“…Each composite DNA extract for the amplicon libraries was amplified separately by PCR in triplicate 50 μl reaction mixtures containing 25 μl 2x GoTaq Green Mastermix (Promega, Madison, WI, USA), 25 μM of each primer and approximately 20 ng template DNA. Amplification was performed using a touchdown PCR program with denaturation at 95°C for 5 min followed by 10 cycles of denaturation at 94°C for 30 s, annealing at 60-50°C for 45 s (−1°C per cycle), and extension at 72°C for 2 min, followed by 30 cycles of 94°C for 30 s, 50°C for 45 s and 72°C for 2 min, with a final 10 min extension step at 72°C (Lentendu et al 2014). The PCR products were separated on a 1.5 % agarose gel and equimolar volumes of the amplified products of the expected size from the three positive replicate amplicons per sample were homogenized.…”