Carbon storage and cycling in boreal forests—the largest terrestrial carbon store—is moderated by complex interactions between trees and soil microorganisms. However, existing methods limit our ability to predict how changes in environmental conditions will alter these associations and the essential ecosystem services they provide. To address this, we developed a metatranscriptomic approach to analyze the impact of nutrient enrichment on Norway spruce fine roots and the community structure, function, and tree–microbe coordination of over 350 root-associated fungal species. In response to altered nutrient status, host trees redefined their relationship with the fungal community by reducing sugar efflux carriers and enhancing defense processes. This resulted in a profound restructuring of the fungal community and a collapse in functional coordination between the tree and the dominant Basidiomycete species, and an increase in functional coordination with versatile Ascomycete species. As such, there was a functional shift in community dominance from Basidiomycetes species, with important roles in enzymatically cycling recalcitrant carbon, to Ascomycete species that have melanized cell walls that are highly resistant to degradation. These changes were accompanied by prominent shifts in transcriptional coordination between over 60 predicted fungal effectors, with more than 5,000 Norway spruce transcripts, providing mechanistic insight into the complex molecular dialogue coordinating host trees and their fungal partners. The host–microbe dynamics captured by this study functionally inform how these complex and sensitive biological relationships may mediate the carbon storage potential of boreal soils under changing nutrient conditions.

The health, growth, and fitness of boreal forest trees are impacted and improved by their associated microbiomes. Microbial gene expression and functional activity can be assayed with RNA sequencing (RNA-Seq) data from host samples. In contrast, phylogenetic marker gene amplicon sequencing data are used to assess taxonomic composition and community structure of the microbiome. Few studies have considered how much of this structural and taxonomic information is included in transcriptomic data from matched samples. Here, we described fungal communities using both host-derived RNA-Seq and fungal ITS1 DNA amplicon sequencing to compare the outcomes between the methods. We used a panel of root and needle samples from the coniferous tree species Picea abies (Norway spruce) growing in untreated (nutrient-deficient) and nutrient-enriched plots at the Flakaliden forest research site in boreal northern Sweden. We show that the relationship between samples and alpha and beta diversity indicated by the fungal transcriptome is in agreement with that generated by the ITS data, while also identifying a lack of taxonomic overlap due to limitations imposed by current database coverage. Furthermore, we demonstrate how metatranscriptomics data additionally provide biologically informative functional insights. At the community level, there were changes in starch and sucrose metabolism, biosynthesis of amino acids, and pentose and glucuronate interconversions, while processing of organic macromolecules, including aromatic and heterocyclic compounds, was enriched in transcripts assigned to the genus Cortinarius. IMPORTANCE A deeper understanding of microbial communities associated with plants is revealing their importance for plant health and productivity. RNA extracted from plant field samples represents the host and other organisms present. Typically, gene expression studies focus on the plant component or, in a limited number of studies, expression in one or more associated organisms. However, metatranscriptomic data are rarely used for taxonomic profiling, which is currently performed using amplicon approaches. We created an assembly-based, reproducible, and hardware-agnostic workflow to taxonomically and functionally annotate fungal RNA-Seq data obtained from Norway spruce roots, which we compared to matching ITS amplicon sequencing data. While we identified some limitations and caveats, we show that functional, taxonomic, and compositional insights can all be obtained from RNA-Seq data. These findings highlight the potential of metatranscriptomics to advance our understanding of interaction, response, and effect between host plants and their associated microbial communities.

Scots pine (Pinus sylvestris L.) is one of the most economically important species to the Swedish forest industry, and cost-efficient planting methods are needed to ensure successful reestablishment after harvesting forest stands. While the majority of clear-cuts are replanted with pre-grown seedlings, direct seeding can be a viable option on poorer sites. Organic fertilizer has been shown to improve planted seedling establishment, but the effect on direct seeding is less well known. Therefore, at a scarified (disc trencher harrowed) clear-cut site in northern Sweden, we evaluated the effect of early, small-scale nitrogen addition on establishment and early recruitment of fungi from the disturbed soil community by site-planted Scots pine seeds. Individual seeds were planted using a moisture retaining germination matrix containing 10 mg nitrogen in the form of either arginine phosphate or ammonium nitrate. After one growing season, we collected seedlings and assessed the fungal community of seedling roots and the surrounding soil. Our results demonstrate that early, small-scale N addition increases seedling survival and needle carbon content, that there is rapid recruitment of ectomycorrhizal fungi to the roots and rhizosphere of the young seedlings and that this rapid recruitment was modified but not prevented by N addition.

Microbial communities are major players in carbon and nitrogen cycling globally and are of particular importance for plant communities in the nutrient poor soils of boreal forests. Especially relevant are the fungal communities in the soil that interact with the plants in multiple ways, indirectly through their pivotal role in the breakdown of organic matter and, more directly, through mycorrhizal symbiosis with plant roots. Large-scale disturbances of these complex microbial communities can lead to shifts in soil carbon storage with unknown and global-scale long-term consequences. To understand the dynamics of these communities and their relationship to associated plants in response to climate change and anthropogenic influence, we need a better understanding of how modern “omics” methods can help us to understand compositional and functional shifts of these microbiomes. Microbial gene expression and functional activity can be assayed with RNA sequencing (RNA-Seq) data from environmental samples. In contrast, currently phylogenetic marker gene amplicon sequencing data is generally used to assess taxonomic composition and community structure of the microbiome. Few studies have considered how much of this structural and taxonomic information is included in RNA-Seq transcriptomic data from matched samples. Here we describe fungal communities using both RNA-Seq and fungal ITS1 DNA amplicon sequencing to compare the outcomes between the methods. We used a panel of root and needle samples from mature stands of the coniferous tree species Picea abies (Norway spruce) growing in untreated (nutrient deficient) and nutrient enriched plots at the Flakaliden forest research site in boreal northern Sweden. We created an assembly-based, reproducible and hardware agnostic workflow to taxonomically and functionally annotate fungal RNA-Seq data obtained from Norway spruce roots, which we compared to matching ITS amplicon sequencing data. We show that the community structure indicated by the fungal transcriptome is in agreement with that generated by the ITS data, while also identifying limitations imposed by current database coverage. Furthermore, we show examples to demonstrate how metatranscriptomics data additionally provides biologically informative functional insight at the community and individual species level. These findings highlight the potential of metatranscriptomics to advance our understanding of interaction, response and effect both between host plants and their associated microbial communities, and among the members of microbial communities in environmental samples in general.