Early microbial colonizers of post-fire soils may exploit either or both PyOM and necromass as a key carbon source. However, relatively little is known about how the metabolism of these respective carbon sources may drive post-fire microbial succession and community recovery. Many microorganisms are able to metabolize polyaromatic compounds with similarities to those found in PyOM, either completely or incompletely . For example, white-rot fungi have been particularly well studied for their ability to metabolize the phenolic polymer lignin. These fungi leverage a combination of peroxidases, laccases, and monooxygenases to initiate the degradation of lignin and other polyaromatic compounds . Non-lignolytic fungi rely primarily on monooxygenases, especially cytochrome P450 monooxygenases, coupled with epoxide hydrolases to initiate the degradation of complex polyaromatic compounds . Several common soil fungi have also been shown to degrade polyaromatic compounds . These fungi include Neurospora crassa, which emerges from burned wood shortly after fire, and Morchella conica, which is a relative of pyrophilous Morchella species that often co-occur with Pyronema species . Fruiting bodies of the genus Pyronema are among the first macrofungi to emerge from burned soil, doing so within weeks to months after fire . There are two currently accepted species of Pyronema: P. domesticum and P. omphalodes ,dutch bucket hydroponic both of which rapidly dominate post-fire fungal communities .
A recent ITS amplicon community analysis showed that Pyronema reads, which made up less than 1% of reads prior to fire achieved a post-fire average relative abundance of 60.34% . Both P. domesticum and P. omphalodes were isolated from fruiting bodies that appeared within months after the catastrophic 2013 Rim Fire in the pine forest of Stanislaus National Forest, which boarders Yosemite National Park . In vitro, Pyronema has a rapid growth rate, but has historically been considered a poor competitor with other soil fungi . Thus, a key question is: what carbon source is used by Pyronema to achieve such high relative abundance post-fire? Does Pyronema simply exploit the available necromass, or do they have the ability to metabolize PyOM as well? Given the dominant status and their early emergence after fire, Pyronema likely play a critical role in the first steps of post-fire succession. Thus, the possibility that Pyronema might contribute to the mineralization of PyOM has far-reaching implications for carbon cycling within post-fire soil communities. In this work, we investigated the hypothesis that early successional pyrophilous fungi such as Pyronema metabolize PyOM. To do so, we measured biomass, sequenced the transcriptome , and measured CO2 efflux from P. domesticum grown on agar media with various carbon sources, including PyOM and burned soil collected from a frequent and high-intensity wildfire site . When grown on media containing burned soil or PyOM, P. domesticum produced significant biomass, activated a diverse suite of cytochrome P450 and FAD-dependent monooxygenases, and comprehensively induced pathways for aromatic substrate utilization. Lastly, we confirmed that P. domesticum mineralized PyOM by measuring CO2 emissions of P. domesticum grown on 13C-labeled PyOM.
Collectively, our results demonstrate the potential for P. domesticum to liberate carbon from PyOM, assimilate it into biomass, and mineralize it to CO2. Thus, pioneering organisms such as P. domesticum may play an important role in the short-term reintegration of PyOM into biologically available carbon in post-fire ecosystems.Pyrogenic organic matter was produced from Pinus strobus wood chips <2 mm at 750°C in a modified Fischer Scientific Lindberg/Blue M Moldatherm box furnace fitted with an Omega CN9600 SERIES Autotune Temperature Controller . We modified the furnace and adapted the PyOM production design developed by Güereña et al. . Briefly, the feedstock was placed in a steel cylinder inside the furnace chamber and subjected to a continuous argon gas supply at a rate of 1 L min−1 to maintain anaerobic conditions during pyrolysis. The heating rate for production of PyOM was kept constant at 5°C min−1 . We held the temperature constant for 30 min once 750°C was reached, after which the PyOM was rapidly cooled by circulating cold water in stainless steel tubes wrapped around the steel cylinder. The PyOM was ground using a mortar and pestle and sieved to collect PyOM with particle size <45 μm.Pyronema domesticum DOB7353 agar plugs were transferred from long-term storage water stocks onto a sucrose agar plate overlaid with cellophane, and incubated at room temperature for ~5–7days. Then, a 6-mm-diameter transfer tube was used to transfer equivalent amounts of mycelium from the initial growth plate to inoculate each experimental agar plate. Cellophane acts as a physical barrier to ensure no agar is transferred to the experimental plates, and to facilitate easy harvesting of all biomass without agar contamination. The integrity of the cellophane is maintained throughout growth, and we never observed physical cellophane degradation after incubation with P. domesticum. Cellulase-encoding genes were annotated in the P. domesticum genome via the CAZy database, and we have included this CAZyme information in the Supplementary Data.
We inoculated P. domesticum hyphae onto four different experimental treatments: 1.5% agar media treatment plates overlaid with cellophane, Vogel’s Minimal Medium agar containing 20 g L−1 sucrose , 10 gL−1 750°C PyOM agar , 10 gL−1 wildfire-burned soil agar , and water agar . Burned soil was collected from 0 to 10 cm in Illilouette Creek Basin via an ethanol-sterilized shovel, and homogenized in plastic zip-top bags. Burned soil was x-ray sterilized , which used a Bremsstrahlung process to generate photons with an energy of 5–7MeV. Both PyOM and soil were added to agar media after autoclaving. Both agar media and cellophane submerged in ddH2O were autoclaved on a liquid cycle at 121°C and ~15–20psi for 30min. Pyronema domesticum was allowed to grow for 4days until it completely covered the plate on each agar media treatment described above . All biomass from each plate was harvested by scraping with a spatula, immediately weighed, and then mixed with 500μl 0.2mM Methylene Blue in a 1.5ml microcentrifuge tube. We adapted Fisher & Sawers’ Methylene Blue biomass quantification protocol . Briefly, tubes of MB-stained biomass were heated at 80°C for 5min, vortexed at maximum speed for 10min, and then heated again at 80°C for 5min. Mycelia were pelleted by centrifugation for 10min at maximum speed in a standard microcentrifuge. About 50μl of the supernatant was combined with 200μl ddH2O and then absorbance was measured at 660nm. Blank wells and wells containing 0.2mMMB were included as controls.Mycelia from sets of three plates were pooled, resulting in three replicate samples for RNA extraction and sequencing. Pooled mycelia were immediately flash frozen with liquid nitrogen. Cells were lysed by beadbeating with 1ml TRIzol . Nucleosomes were removed by gently shaking for 5min at room temperature. About 200 μl chloroform was added, briefly bead-beaten, and then centrifuged to pellet cell debris. The aqueous phase was then used for RNA purification with the Zymo Direct-zol RNA MiniPrep kit . The qb3 facility at University of California, Berkeley, quantified RNA quality and concentration via Bioanalyzer and then carried out library preparation and sequencing on an Illumina NovaSeq 6000 Platform.Raw reads were manually inspected for quality using FastQC v0.11.5 and then trimmed and quality filtered with Trimmomatic v0.36 . HISAT2 v2.1.0 mapped quality reads to the P. domesticum DOB7353 v1.0 genome . Raw counts per gene were generated with HTSeq v0.9.1 . Raw counts were normalized, a principal component analysis plot was generated, and differential expression was calculated with DESeq2 v1.24.0 on R v3.6.1 . To determine whether expression profiles were significantly different across treatments,dutch buckets system we used PERMANOVA from the adonis function from the vegan package v2.5-7 . Functional gene annotations were downloaded from the Joint Genome Institute’s Mycocosm portal . To test the transcriptional response of P. domesticum to fireaffected substrates, we grew P. domesticum on two agar media treatments containing burned substrates and on two control agar media treatments. Both burned treatments shared similarities with the severely burned pine forest in the Sierra Nevada from which P. domesticum was originally isolated. These burned substrates were 750°C P. strobus wood PyOM and wildfire burned soil that was collected near the original isolation site of P. domesticum. We observed distinct differences in the macroscopic growth pattern of P. domesticum when grown on the four different agar media treatments; PyOM, burned soil, sucrose, and water agar . After inoculating agar treatment plates with equivalent amounts of mycelia and incubating for 4days, a substantial amount of biomass was produced on sucrose . Growth on PyOM and, to a lesser extent, burned soil both produced an intermediate amount of biomass. Notably, P. domesticum has a tufted or fluffy macroscopic morphology on sucrose and to a lesser extent, PyOM. Lastly, there was observable growth on water agar, but biomass production was minimal . These data illustrate that the sucrose and water treatments are functional experimental controls for robust growth and minimal growth, respectively.
After 4days of growth on each substrate, the biomass from each treatment was harvested, and RNA was extracted for sequencing. Across PC2 , the transcriptomes from the water and sucrose conditions were at opposite ends, while transcriptomes from the PyOM and burned soil were located at an intermediate point near the origin. A possible explanation for this distribution is that PC2 describes the overall amount of bioavailable carbon and other nutrients. Water agar representing starvation contains the least amount of nutrients, the PyOM and soil containing intermediate amounts, and sucrose agar containing the most. Across PC1 , the PyOM-associated transcriptomes were located at one end of the axis, while transcriptomes from water and sucrose conditions were at the opposite end. The burned soil transcriptomes were at an intermediate position, closer to the sucrose and water conditions than PyOM. One possibility is that PC1 reflects the amount of PyOM present in the medium, since the PyOM medium contained the most, burned soil contained less, and sucrose and water media lacked any at all. Together, these results indicate that the transcriptional response of P. domesticum to burned or pyrolyzed substrates is significantly different from the response to water or sucrose, and the response to PyOM is particularly distinct.Compared to sucrose, on water agar we observed significant upregulation of 318 genes , including 31 transporters and 86 genes involved in the metabolism of diverse substrates, including the catabolism of amino acids and nucleotides . Several general stress response genes were also induced on water agar compared to sucrose; specifically, seven different heat shock proteins and two proteins involved in programmed cell death. Surprisingly, invertase, the enzyme that hydrolyzes sucrose, was not significantly downregulated on water compared to sucrose . In contrast to the 318 genes that were upregulated on water compared to sucrose, there were only 94 genes significantly upregulated on sucrose compared to water, including a sugar/hydrogen symporter, and 23 genes involved in primary metabolism, biosynthesis, and development . Taken together, these data demonstrate that growth on water agar induces a stress response program that includes genes involved in catabolism of macromolecules and scavenging for alternative nutrient sources. In contrast, growth on sucrose allows for a more streamlined transcriptome focused on growth powered by the metabolism of simple sugars.To examine the nutritional and metabolic response to burned or pyrolyzed substrates, we calculated differential expression of genes in each treatment compared to sucrose and used functional gene annotations to categorize genes that were significantly upregulated at least 4-fold . We observed the largest shift in gene expression on 750°C PyOM with a total of 519 significantly upregulated genes . Around 227 genes were upregulated on burned soil, and the majority of those overlapped with genes induced on PyOM and/or water . We note that invertase was significantly downregulated on PyOM compared to sucrose , and to a lesser extent on soil compared to sucrose . The 171 genes that were induced on water and at least one of the two substrates containing PyOM characterized a stress response associated with decreased nutrient availability. Among these 171 genes are 19 transporters and four general stress response genes including two heat shock proteins . We observed signatures of nitrogen stress in the water, PyOM, and soil conditions compared to sucrose minimal medium, which contains ammonium nitrate as a nitrogen source. These putative nitrogen stress-responsive genes include genes involved in ammonium production, nitrogen metabolism, and a putative ortholog of the conserved Aspergillus nidulans transcription factor TamA . Lastly, the gene_6383 encodes a putative laccase and was upregulated 26–60 fold on water, soil, and PyOM, compared to sucrose.