Effects of nitrogen deposition on carbon and nutrient cycling along a natural soil acidity gradient as revealed by metagenomics.

Nitrogen (N) deposition and soil acidification are environmental challenges affecting ecosystem functioning, health, and biodiversity, but their effects on functional genes are poorly understood. Here, we utilized metabarcoding and metagenomics to investigate the responses of soil functional genes to N deposition along a natural soil pH gradient. Soil N content was uncorrelated with pH, enabling us to investigate their effects separately. Soil acidity strongly and negatively affected the relative abundances of most cluster of orthologous gene categories of the metabolism supercategory. Similarly, soil acidity negatively affected the diversity of functional genes related to carbon and N but not phosphorus cycling. Multivariate analyses showed that soil pH was the most important factor affecting microbial and functional gene composition, while the effects of N deposition were less important. Relative abundance of KEGG functional modules related to different parts of the studied cycles showed variable responses to soil acidity and N deposition. Furthermore, our results suggested that the diversity-function relationship reported for other organisms also applies to soil microbiomes. Since N deposition and soil pH affected microbial taxonomic and functional composition to a different extent, we conclude that N deposition effects might be primarily mediated through soil acidification in forest ecosystems.

Regional-Scale In-Depth Analysis of Soil Fungal Diversity Reveals Strong pH and Plant Species Effects in Northern Europe.

Soil microbiome has a pivotal role in ecosystem functioning, yet little is known about its build-up from local to regional scales. In a multi-year regional-scale survey involving 1251 plots and long-read third-generation sequencing, we found that soil pH has the strongest effect on the diversity of fungi and its multiple taxonomic and functional groups. The pH effects were typically unimodal, usually both direct and indirect through tree species, soil nutrients or mold abundance. Individual tree species, particularly Pinus sylvestris, Picea abies, and Populus x wettsteinii, and overall ectomycorrhizal plant proportion had relatively stronger effects on the diversity of biotrophic fungi than saprotrophic fungi. We found strong temporal sampling and investigator biases for the abundance of molds, but generally all spatial, temporal and microclimatic effects were weak. Richness of fungi and several functional groups was highest in woodlands and around ruins of buildings but lowest in bogs, with marked group-specific trends. In contrast to our expectations, diversity of soil fungi tended to be higher in forest island habitats potentially due to the edge effect, but fungal richness declined with island distance and in response to forest fragmentation. Virgin forests supported somewhat higher fungal diversity than old non-pristine forests, but there were no differences in richness between natural and anthropogenic habitats such as parks and coppiced gardens. Diversity of most fungal groups suffered from management of seminatural woodlands and parks and thinning of forests, but especially for forests the results depended on fungal group and time since partial harvesting. We conclude that the positive effects of tree diversity on overall fungal richness represent a combined niche effect of soil properties and intimate associations.

Relative Performance of MinION (Oxford Nanopore Technologies) versus Sequel (Pacific Biosciences) Third-Generation Sequencing Instruments in Identification of Agricultural and Forest Fungal Pathogens.

Culture-based molecular identification methods have revolutionized detection of pathogens, yet these methods are slow and may yield inconclusive results from environmental materials. The second-generation sequencing tools have much-improved precision and sensitivity of detection, but these analyses are costly and may take several days to months. Of the third-generation sequencing techniques, the portable MinION device (Oxford Nanopore Technologies) has received much attention because of its small size and possibility of rapid analysis at reasonable cost. Here, we compare the relative performances of two third-generation sequencing instruments, MinION and Sequel (Pacific Biosciences), in identification and diagnostics of fungal and oomycete pathogens from conifer (Pinaceae) needles and potato (Solanum tuberosum) leaves and tubers. We demonstrate that the Sequel instrument is efficient for metabarcoding of complex samples, whereas MinION is not suited for this purpose due to a high error rate and multiple biases. However, we find that MinION can be utilized for rapid and accurate identification of dominant pathogenic organisms and other associated organisms from plant tissues following both amplicon-based and PCR-free metagenomics approaches. Using the metagenomics approach with shortened DNA extraction and incubation times, we performed the entire MinION workflow, from sample preparation through DNA extraction, sequencing, bioinformatics, and interpretation, in 2.5 h. We advocate the use of MinION for rapid diagnostics of pathogens and potentially other organisms, but care needs to be taken to control or account for multiple potential technical biases.IMPORTANCE Microbial pathogens cause enormous losses to agriculture and forestry, but current combined culturing- and molecular identification-based detection methods are too slow for rapid identification and application of countermeasures. Here, we develop new and rapid protocols for Oxford Nanopore MinION-based third-generation diagnostics of plant pathogens that greatly improve the speed of diagnostics. However, due to high error rate and technical biases in MinION, the Pacific BioSciences Sequel platform is more useful for in-depth amplicon-based biodiversity monitoring (metabarcoding) from complex environmental samples.

Novel soil-inhabiting clades fill gaps in the fungal tree of life.

BACKGROUND: Fungi are a diverse eukaryotic group of degraders, pathogens, and symbionts, with many lineages known only from DNA sequences in soil, sediments, air, and water. RESULTS: We provide rough phylogenetic placement and principal niche analysis for >40 previously unrecognized fungal groups at the order and class level from global soil samples based on combined 18S (nSSU) and 28S (nLSU) rRNA gene sequences. Especially, Rozellomycota (Cryptomycota), Zygomycota s.lat, Ascomycota, and Basidiomycota are rich in novel fungal lineages, most of which exhibit distinct preferences for climate and soil pH. CONCLUSIONS: This study uncovers the great phylogenetic richness of previously unrecognized order- to phylum-level fungal lineages. Most of these rare groups are distributed in different ecosystems of the world but exhibit distinct ecological preferences for climate or soil pH. Across the fungal kingdom, tropical and non-tropical habitats are equally likely to harbor novel groups. We advocate that a combination of traditional and high-throughput sequencing methods enable efficient recovery and phylogenetic placement of such unknown taxonomic groups.