Perturbance and Stimulation : using Nitrogen Addition and High-Throughput Sequencing to Study Fungal Communities in Boreal Forests

Sammanfattning: Fungal communities are major players in globally important nutrient cycling processes, and form symbioses with most terrestrial plants. In the nitrogen (N) limited Swedish boreal forest, ectomycorrhizal (EcM) fungi colonize most roots of the economically important and stand dominating conifer species, Norway spruce (Picea abies) and Scots pine (Pinus sylvestris), with significant implications for tree nutrition and decomposition processes. Long-term sustainable forestry practices require a deeper understanding of biotic and abiotic factors influencing forest health and tree growth. While high-throughput sequencing technologies such as DNA amplicon sequencing or RNA-based metatranscriptomics have furthered our understanding of fungal communities, there are still many details of EcM symbiosis and decomposition processes that we do not understand. In this thesis, I have used these sequencing methods to further our understanding of fungal communities in the boreal forest, and how they are influenced by forest management and N addition. In the first part of this thesis, I investigated how early fertilization of seeded or planted seedlings affects seedling growth and survival, and the fungal communities associated with the growing seedlings, assessed by amplicon sequencing. In two manuscripts I show that seeds or seedlings planted into scarified clearcut soil are rapidly colonized by site indigenous fungi, including many EcM species. I show that small doses of added N increase survival of sown seeds and that organic N (in the form of arginine) can increase early root growth of planted seedlings. This light fertilization did not perturb early fungal community succession. In the second part, I co-developed a workflow for de novo assembly as well as functional and taxonomic annotation of complex fungal community RNA sequencing data, in order to advance our ability to utilize metatranscriptomics (not only) as an alternative to DNA amplicon approaches. I assessed the outcome of this workflow by comparison to the currently most widely employed method of DNA amplicon sequencing, finding that both methods provide highly congruent insights into among-sample relationships and alpha and beta diversity. I then demonstrated use of the functional annotation of the metatranscriptomic data to provide biological insight into fungal community responses to high levels of N addition. It is known that N addition to boreal forests, apart from stimulating tree growth, perturbs the natural, N-limited status and leads to significant changes in fungal community composition and soil chemistry. Using metatranscriptomic data and the newly designed workflow enabled us to test the hypothesis that N addition can inhibit decomposition in cold climates, at least in part, by rendering the oxidative enzymes used for so-called “white rot” ligninolytic decay energetically uncompetitive. Moreover, in a study using transcriptomic data from Norway spruce roots and the associated EcM fungi, we show that N addition leads to a reprogramming of the mycorrhizal symbiosis controlled by the tree, thus favoring fungal species that have previously been described as N tolerant. In general, high-throughput sequencing methods have furthered our understanding of fungal community dynamics, and this thesis provides a powerful new part of the toolbox for studying these highly complex systems and contributes new perspectives to our knowledge of how fungal communities respond to N addition and forest management, from the perspective of soil biochemical processes and the EcM symbiosis.