Effects of Bio-Ash Amendments on the Metabolism of Ectomycorrhizal Fungi: A Method Development and Metabolomic Study

Forest ecosystems have played a fundamental role in the development of our society. Since the beginning of the civilization, forest have provided us with wood as a product for construction, tools, furniture and domestic heating. The well-being of the forest is therefore fundamental to our existence. Today, our growing societies have increased energy needs; the resulting depletion of fossil reserves and the effects of their use has again shown how the forest is among the most important alternatives for sustainability of our ecosystem. In order to responsibly make this resource a key part of our energy and material supply, we need to understand how forestry practices influence the different processes taking place in the forest ecosystems. The use of raw material from forest as energy source produces huge amounts of ash. The ash contains the base cations that have once been translocated from soil to the upper parts of the trees. Ash recycling has therefore been suggested as a measure to counteract soil acidification due to extensive harvest. Since spreading of ash can have great effects on the forest, it is important to understand which these effects are and how big they might be. This thesis focuses on the effects that such an ash recycling may have on the metabolism of ectomycorrhizal fungi; that is, fungi that are able to colonize root of trees, and contribute to the acquisition of nutrients and water from soil. The work presented here utilized an in vitro metabolomic approach on eight species of ectomycorrhizal fungi normally found in boreal forests. A targeted metabolomic study addressed the effects of ash amendments on growth, external pH and the exudation of low molecular mass organic acids, amino acids and hydroxamate siderophores. This was complemented by an untargeted metabolomic study to address the effects of ash amendment on the general metabolism of the fungal species. Analyses were performed with well-established chemical methods, and some that had to be developed specifically for this thesis work. A method for the analysis of amino acids without derivatization and yet compatible with mass spectrometry had to be developed and validated. The result was a robust method that works well with external calibration, shows good long-term stability, relatively low detections limits and high sample throughput. A screening protocol for the determination of siderophores from mass spectrometry data was also established. The metabolomic studies showed that b