Evaluation of metabarcoding primers for analysis of soil nematode communities

While recent advances in next-generation sequencing technologies have accelerated research in microbial ecology, the application of high throughput approaches to study the ecology of nematodes remains unresolved due to several issues, e.g., whether to include an initial nematode extraction step or not, the lack of consensus on the best performing primer combination, and the absence of a curated nematode reference database. The objective of this method development study was to compare different primer sets to identify the most suitable primer set for the metabarcoding of nematodes without initial nematode extraction. We tested four primer sets for amplicon sequencing: JB3/JB5 (mitochondrial, I3-M11 partition of COI gene), SSU_04F/SSU_22R (18S rRNA, V1-V2 regions), and Nemf/18Sr2b (18S rRNA, V6-V8 regions) from earlier studies, as well as MMSF/MMSR (18S rRNA, V4-V5 regions), a newly developed primer set. We used DNA from 22 nematode taxa, 10 mock communities, 20 soil samples, 4 root samples, and one bulk soil. We amplified the target regions from the DNA samples with the four different primer combinations and sequenced the amplicons on an Illumina MiSeq sequencing platform. We found that the Nemf/18Sr2b primer set was superior for detecting soil nematodes compared to the other primer sets based on our sequencing results and on the annotation of our sequence reads at the genus and species ranks. This primer set generated 74% reads of Nematoda origin in the soil samples. Additionally, this primer set did well with the mock communities, detecting all the included specimens. It also worked better in the root samples than the other primer set that was tested. Therefore, we suggest that the Nemf/18Sr2b primer set could be used to study rhizosphere soil and root associated nematodes, and this can be done without an initial nematode extraction step

Enhanced priming of old, not new soil carbon at elevated atmospheric CO2

Rising atmospheric CO2 concentrations accompanied by global warming and altered precipitation patterns calls for assessment of long-term effects of these global changes on carbon (C) dynamics in terrestrial ecosystems, as changes in net C exchange between soil and atmosphere will impact the atmospheric CO2 concentration profoundly. In many ecosystems, including the heath/grassland system studied here, increased plant production at elevated CO2 increase fresh C input from litter and root exudates to the soil and concurrently decrease soil N availability. Supply of labile C to the soil may accelerate the decomposition of soil organic C (SOC), a phenomenon termed ‘the priming effect’, and the priming effect is most pronounced at low soil N availability. Hence, we hypothesized that priming of SOC decomposition in response to labile C addition would increase in soil exposed to long-term elevated CO2 exposure. Further, we hypothesized that long-term warming would enhance SOC priming rates, whereas drought would decrease the priming response. We incubated soil from a long-term, full-factorial climate change field experiment, with the factors elevated atmospheric CO2 concentration, warming and prolonged summer drought with either labile C (sucrose) or water to assess the impact of labile C on SOC dynamics. We used sucrose with a 13C/12C signature that is distinct from that of the native SOC, which allowed us to assess the contribution of these two C sources to the CO2 evolved. Sucrose induced priming of SOC, and the priming response was higher in soil exposed to long-term elevated CO2 treatment. Drought tended to decrease the priming response, whereas long-term warming did not affect the level of priming significantly. We were also able to assess whether SOC-derived primed C in elevated CO2 soil was assimilated before or after the initiation of the CO2 treatment 8 years prior to sampling, because CO2 concentrations were raised by fumigating the experimental plots with pure CO2 that was 13C-depleted compared to ambient CO2. Surprisingly, we conclude that sucrose addition primed decomposition of relatively old SOC fractions, i.e. SOC assimilated more than 8 years before sampling

The "soil microbial loop" is not always needed to explain protozoan stimulation of plants

Protozoa stimulate plant growth, but we do not completely understand the underlying mechanisms, and different hypotheses seek to explain this phenomenon. To test these hypotheses, we grew the grass Yorkshire fog (Holcus lanatus) in pots with soil, which contained either (1) no organisms but bacteria – or (2) bacteria and protozoa. Half of the pots received a glucose treatment so as to mimic an additional root exudation.We measured plant growth and plant nitrogen uptake, along with various microbial pools and processes that support plant growth. Protozoan presence significantly enhanced soil nitrogen mineralization, plant nitrogen uptake from organic nitrogen sources, plant nitrogen content, and plant growth. By contrast, we found no evidence that glucose addition, mimicking root exudation, increased soil nitrogen availability and plant nitrogen uptake. Moreover, although protozoan presence affected bacterial community structure, it did not affect the proportion of IAA-producing bacteria in the community or plant root morphology. These results refute the ''soil microbial loop'' hypotheses, which suggest that protozoan stimulation of plant growth results from complex interactions between plant roots, bacteria and protozoa. Our experiment thus favours the simple explanation that increased nitrogen availability is the key factor behind the positive protozoan effect on plant growth. To exploit natural resources in an efficient and environmentally friendly way, we need to understand in detail the functioning of ecosystems. This study stresses that to achieve this, it is still urgent, besides investigating intricate food-web and signal compound interactions, also to focus on the basic stoichiometric and energetic aspects of organisms. (Résumé d'auteur