Pyrolyzed Substrates Induce Aromatic Compound Metabolism in the Post-fire Fungus, Pyronema domesticum

Abstract

Wildfires represent a fundamental and profound disturbance in many ecosystems, and their frequency and severity are increasing in many regions of the world. Fire affects soil by removing carbon in the form of CO₂ and transforming remaining surface carbon into pyrolyzed organic matter (PyOM). Fires also generate substantial necromass at depths where the heat kills soil organisms but does not catalyze the formation of PyOM. Pyronema species strongly dominate soil fungal communities within weeks to months after fire. However, the carbon pool (i.e., necromass or PyOM) that fuels their rise in abundance is unknown. We used a Pyronema domesticum isolate from the catastrophic 2013 Rim Fire (CA, United States) to ask whether P. domesticum is capable of metabolizing PyOM. Pyronema domesticum grew readily on agar media where the sole carbon source was PyOM (specifically, pine wood PyOM produced at 750°C). Using RNAseq, we investigated the response of P. domesticum to PyOM and observed a comprehensive induction of genes involved in the metabolism and mineralization of aromatic compounds, typical of those found in PyOM. Lastly, we used ¹³C-labeled 750°C PyOM to demonstrate that P. domesticum is capable of mineralizing PyOM to CO₂. Collectively, our results indicate a robust potential for P. domesticum to liberate carbon from PyOM in post-fire ecosystems and return it to the bioavailable carbon pool.

Keywords: PyOM; RNAseq; aromatic hydorcarbons; carbon metabolic activity; filamenous fungi; fire.

Figure 1

Pyronema growth on natural and laboratory substrates. (A) Pyronema sp. fruiting on burned soil 6months after the 2018 Camp Fire began near Paradise, California, United States. (B) Pyronema domesticum DOB7353 ascocarps on the edge of a water agar plate. (C) Pyronema domesticum DOB7353 growing on four different agar media treatments: water agar with no added nutrients (“Water”), wildfire-burned soil collected near the original isolation site for P. domesticum DOB7353 (“Soil”), 750°C White Pine wood char [“pyrolyzed organic matter (PyOM)”], or Vogel’s minimal medium with sucrose as a carbon source (“Sucrose”). All plates were inoculated in the center of the plate (bottom-right corner of photo) with mycelium from a 6-mm-diameter punch of an actively growing P. domesticum colony. Scale bar=1cm. (D) Average amount of P. domesticum DOB7353 biomass on one full plate as show in (C), quantified by measuring the amount of Methylene Blue (MB) stain remaining after absorption by P. domesticum hyphae. All treatments are significantly different from each other, lowercase letters above each bar indicate significantly different groups (ANOVA+Tukey’s test, p<0.0001, n=5, error bars=SD). The “No hyphae” control is pure 0.2mMMB without any biomass treatment. The “No MB” control is a blank well.

Figure 2

Pyrolyzed substrates induce expression of distinct sets of genes in P. domesticum. Principal Component Analysis (PCA) plot illustrating the variation between each sample transcriptome (normalized expression values). Prior to RNA extraction, P. domesticum DOB7353 was grown in triplicate on four different agar media treatments.

Figure 3

Pyrolyzed substrates induce expression of genes involved in stress response and PyOM metabolism. (A) Venn diagram showing the number of significantly upregulated genes in each treatment compared to sucrose (adjusted value of p<0.01, fold change>4, n=3). (B) Number of significantly upregulated genes compared to expression on sucrose in each functional gene category (adjusted value of p<0.01, fold change>4, n=3). Functional gene categories were determined via KEGG, GO, and pfam annotations. Stacked black and orange bars indicate the number of genes upregulated on PyOM alone (black) or the overlap between PyOM and soil (orange and black). We defined stress-response genes as those which are upregulated on water agar. Blue bars indicate the number of genes that are upregulated on both water and burned or pyrolyzed substrates for each functional category.

Figure 4

Metabolic map highlighting aromatic compound metabolism induced by growth on pyrolyzed substrates. Significantly upregulated genes mapped onto the canonical pathways for aromatic compound metabolism (adjusted value of p<0.01, fold change>2, n=3). Bolded arrows indicate a fold change>8 on PyOM compared to sucrose. Each gene is indicated as a black-outlined box, and the proteins encoded by these genes are indicated as purple text. The color fill of the box indicates the condition(s) in which the gene was upregulated. Multi-colored boxes are slightly larger than mono-color boxes to increase visibility of the colors and to highlight genes that are induced in more than one condition. Diagonal parallel lines within a box and associated dashed lines indicate genes that were expressed, but not differentially expressed under the tested conditions.

Figure 5

Cumulative mean CO₂ emissions from P. domesticum growing on ¹³C-labeled 750°C PyOM. (A) Mean cumulative CO₂ measured over time from the enclosed headspace of jars containing either sterile (uninoculated, red diamonds) 750°C PyOM agar, or identical plates inoculated with P. domesticum (inoculated, dark grey squares; n=5, error bars=SE). (B) Mean cumulative CO₂ from P. domesticum inoculated jars, normalized by the uninoculated controls, and then partitioned into PyOM-derived C (black squares) and non-PyOM-derived C (light grey squares) using ¹³C partitioning (n=5, error bars=SE).