This method employs liquid chromatography-tandem mass spectrometry to rapidly quantify chitin-derived glucosamine for estimating fungal biomass. Analyte retention was achieved using hydrophilic interaction liquid chromatography, with a zwitter-ionic stationary phase (ZIC-HILIC), and isocratic elution using 60% 5 mM ammonium formate buffer (pH 3.0) and 40% ACN. Inclusion of muramic acid and its chromatographic separation from glucosamine enabled calculation of the bacterial contribution to the latter. Galactosamine, an isobaric isomer to glucosamine, found in significant amounts in soil samples, was also investigated. The two isomers form the same precursor and product ions and could not be chromatographically separated using this rapid method. Instead, glucosamine and galactosamine were distinguished mathematically, using the linear relationships describing the differences in product ion intensities for the two analytes. The m/z transitions of 180 → 72 and 180 → 84 were applied for the detection of glucosamine and galactosamine and that of 252 → 126 for muramic acid. Limits of detection were in the nanomolar range for all included analytes. The total analysis time was 6 min, providing a high sample throughput method.

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A liquid chromatography with electrospray ionization mass spectrometry method was developed to quantitatively and qualitatively analyze 13 hydroxamate siderophores (ferrichrome, ferrirubin, ferrirhodin, ferrichrysin, ferricrocin, ferrioxamine B, D1 , E and G, neocoprogen I and II, coprogen and triacetylfusarinine C). Samples were preconcentrated on-line by a switch-valve setup prior to analyte separation on a Kinetex C18 column. Gradient elution was performed using a mixture of an ammonium formate buffer and acetonitrile. Total analysis time including column conditioning was 20.5 min. Analytes were fragmented by applying collision-induced dissociation, enabling structural identification by tandem mass spectrometry. Limit of detection values for the selected ion monitoring method ranged from 71 pM to 1.5 nM with corresponding values of two to nine times higher for the multiple reaction monitoring method. The liquid chromatography with mass spectrometry method resulted in a robust and sensitive quantification of hydroxamate siderophores as indicated by retention time stability, linearity, sensitivity, precision and recovery. The analytical error of the methods, assessed through random-order, duplicate analysis of soil samples extracted with a mixture of 10 mM phosphate buffer and methanol, appears negligible in relation to between-sample variations.

Environmental contextSpreading recycled wood ash in forests may counteract acidification and nutrient losses, but the process may also affect symbiotic fungi in these eco-systems. We show how fungal species react when exposed to ash solutions; for example, by an increased release of organic acids and other compounds. These effects can influence pH and metal availability in forest soils treated with ash. AbstractRecycling of wood ash may counteract acidification and losses of base cations resulting from whole-tree harvesting in boreal forest ecosystems. The effects of ash treatment on growth and exudation of eight ectomycorrhizal fungal species were investigated in this study. Six basidiomycetes and two ascomycetes were grown in liquid pure culture with different levels of ash amendments. Biomass production, pH and the exudation of 17 low-molecular-mass organic acids (LMMOAs), 23 amino acids (AAs) and 9 hydroxamate siderophores (HSs) were recorded after 1, 2 and 4 weeks of incubation. Ash did not affect fungal growth, but resulted in higher exudation of the investigated compounds, in particular LMMOAs. Ash also influenced the composition of the exudates. We measured exudation of LMMOAs and AAs up to millimolar and micromolar concentrations respectively. For example, Rhizopogon roseolus mainly produced oxalic acid, whereas Lactarius rufus and Tomentellopsis submollis produced the highest concentrations of AAs. Ferricrocin, the only HS detected, was exuded at the nanomolar level. Exudation responses were also highly species-dependent, e.g. the ascomycetous isolates that produced the largest biomass released low amounts of exudates compared with the basidiomycetes, and were the only ones producing siderophores. This growth–exudation response to ash is likely a trade-off in carbon allocation whereby the mycorrhizal fungal species invest carbon in either higher biomass production or higher exudation.