Assembly of root-associated N2O-reducing communities of annual crops is governed by selection for nosZ clade I over clade II

Abstract

The rhizosphere is a hotspot for denitrification. The nitrous oxide (N2O) reductase among denitrifiers and non-denitrifying N2O reducers is the only known N2O sink in the biosphere. We hypothesized that the composition of root-associated N2O-reducing communities when establishing on annual crops depend on soil type and plant species, but that assembly processes are independent of these factors and differ between nosZ clade I and II. Using a pot experiment with barley and sunflower and two soils, we analyzed the abundance, composition and diversity of soil and root-associated N2O reducing communities by qPCR and amplicon sequencing of nosZ. Clade I was more abundant on roots compared to soil, while clade II showed the opposite. In barley, this pattern coincided with N2O availability, determined as potential N2O production rates, but for sunflower no N2O production was detected in the root compartment. Root and soil nosZ communities differed in composition and phylogeny-based community analyses indicated that assembly of root-associated N2O reducers was driven by the interaction between plant and soil type, with inferred competition being more influential than habitat selection. Selection between clades I and II in the root/soil interface is suggested, which may have functional consequences since most clade I microorganisms can produce N2O.

Targeted metagenomics using probe capture detect a larger diversity of nitrogen and methane cycling genes in complex microbial communities than traditional metagenomics

“…The copyright holder for this preprint this version posted November 4, 2022. ; https://doi.org/10. 1101/2022 Comparison between agricultural and wetland soils showed that targeted metagenomics detected substantially higher diversity of archaeal amoA genes than PCR amplicon sequencing, while, as expected, traditional metagenomics detected a lower diversity than either of those two methods (Fig. 4A).…”

“…A reference phylogeny and alignment for nosZ was generated from full length nosZ amino acid sequences obtained from genomes downloaded from the NCBI genome database (accessed October 2019). A profile HMM for nosZ was generated from a previously published nosZ dataset (Graf et al, 2022), and then used to search translated open reading frames in downloaded genomes (e-value = 0.0001). Hits were then dereplicated at 99% amino acid similarity and aligned by amino acid using hmmalign within the HMMer suite (v.3.1.2).…”

“…Phylogenetic placement of nosZ reads obtained from agricultural soil using A) shotgun metagenomics and B) targeted metagenomics. The reference phylogeny (Graf et al, 2022) is based on amino acid sequences analyzed using the LG+R10 substitution model in IQ-TREE, and node symbols indicate the location of placements in the reference tree. Symbol size corresponds to the relative abundance of reads placed at each node, and the scale bar at center indicates branch length in the reference tree.…”

“…In order to compare the diversity of specific functional genes detected by targeted metagenomics, traditional metagenomics, and PCR amplicon sequencing, we used the archaeal amoA gene as a test case, for which there is a comprehensive reference database for classification available (Alves et al, 2018). Additionally, we compared the detection of nosZ genes between the targeted and untargeted metagenomics approaches in the agricultural soil, also based on an existing reference database (Graf et al, 2022). Comparisons were performed on different levels between: (i) targeted and traditional metagenomes, and PCR amplicons of the archaeal amoA gene in the agricultural soil (Fig.…”

“…The copyright holder for this preprint this version posted November 4, 2022. ; https://doi.org/10. 1101/2022 To improve the analysis of critical, but low abundant microbial guilds involved in N and CH4 cycling within natural communities, a probe-based targeted metagenomics approach for key genes involved in these processes could be advantageous . For eukaryotes, targeted, high-throughput sequencing approaches known as probe capture, also called hybridisation capture or hybridisation-based target enrichment, have been used to study complex eukaryotic samples such as human exons, ancient human genomes, plant transcriptomes, and cancer marker single nucleotide polymorphisms (Skoglund et al 2014, Ichida et al 2019, Futema et al 2012, Kamil et al 2021, Bewicke-Copley et al 2019.…”