Regulation of fungal decomposition at single-cell level

Abstract

Filamentous fungi play a key role as decomposers in Earth's nutrient cycles. In soils, substrates are heterogeneously distributed in microenvironments. Hence, individual hyphae of a mycelium may experience very different environmental conditions simultaneously. In the current work, we investigated how fungi cope with local environmental variations at single-cell level. We developed a method based on infrared spectroscopy that allows the direct, in-situ chemical imaging of the decomposition activity of individual hyphal tips. Colonies of the ectomycorrhizal Basidiomycete Paxillus involutus were grown on liquid media, while parts of colonies were allowed to colonize lignin patches. Oxidative decomposition of lignin by individual hyphae growing under different conditions was followed for a period of seven days. We identified two sub-populations of hyphal tips: one with low decomposition activity and one with much higher activity. Active cells secreted more extracellular polymeric substances and oxidized lignin more strongly. The ratio of active to inactive hyphae strongly depended on the environmental conditions in lignin patches, but was further mediated by the decomposition activity of entire mycelia. Phenotypic heterogeneity occurring between genetically identical hyphal tips may be an important strategy for filamentous fungi to cope with heterogeneous and constantly changing soil environments.

Fig. 1. Schematic overview of the experimental set-up and data collection routine used in chemical imaging of the oxidative decomposition of lignin by individual hyphal tips of Paxillus involutus.

Starting from the top left image and following clockwise: a Schematic overview of the experimental set-up where P. involutus cultures were grown on liquid media and allowed to colonize two lignin patches (one patch containing no iron, “−Fe”, and one patch containing 1 mg Fe g⁻¹ lignin, “+Fe”). The liquid medium either contained 74 µM FeCl₃•6H₂O (experiment 1, “+Fe”, resulting in mycelia with high oxidative decomposition activity) or no iron (experiment 2, “−Fe”, resulting in mycelia with low oxidative decomposition activity). b Six individual hyphal tips were imaged per lignin patch, with the white light image of only one hyphal tip shown in this figure as an example. c Pixels in hyperspectral images were clustered according to chemical similarity using in-house built software. d Subsequently, the average spectrum of pixels not affected by decomposition (red, background) was subtracted from the average spectrum of pixels affected by oxidative decomposition (blue, hypha), resulting in a single difference spectrum (black) for each image. This difference spectrum represents the average net effect of metabolic activity (including secretion of metabolic products and oxidative decomposition of the lignin substrate) caused by a single hypha. Absorbance values are expressed in arbitrary units (a.u.). e Finally, similarities between difference spectra were visualized using principal component analyses and statistically compared across incubation times, lignin patches and experiments using permutational multivariate analysis of variance.

Fig. 2. Regulation of the metabolic activity of individual hyphal tips of P. involutus by the presence or absence of iron in lignin patches or liquid incubation media.

Principal component analysis was used to visually compare difference spectra across incubation times, patch types and experiments. a The first two principal components of difference spectra marked according to incubation time (days). b The first two principal components of difference spectra marked according to lignin patch type. “Lignin −Fe” denotes bare lignin patches; “Lignin+Fe” denotes lignin patches containing 1 mg Fe g⁻¹ lignin in the form of ferrihydrite. c The first two principal components of difference spectra marked according to experiment. In experiment 1, liquid media contained 74 µM FeCl₃•6H₂O, resulting in mycelia with high overall oxidative decomposition activity, whereas in experiment 2, liquid media did not contain iron, resulting in mycelia with low overall oxidative decomposition activity. d The first two principal components of difference spectra marked according to clusters identified using model-based clustering. e Average difference spectra for each of the two clusters identified with model-based clustering. Lines above and below each measured wavenumber represent standard deviations (n = 92 for cluster 1 and n = 76 for cluster 2). Absorbance values equal to 0 indicate no difference in absorbance between hypha spectrum and background spectrum. Positive absorbance values indicate relative increases in absorbance values caused by the presence of hyphae and negative values indicate relative decreases in absorbance values caused by oxidative decomposition of lignin. In the inset, the spectral region from 1480 cm⁻¹ to 1520 cm⁻¹ is enlarged. Error bars were removed from the inset for clarity. Negative absorbance values in this range are indicative for oxidative lignin decomposition. Absorbance values are expressed in arbitrary units (a.u.). f Summary of the number of hyphae with high or low metabolic activity for each patch type and experiment.