Gene_6383 was the only laccase gene that was identified in the P. domesticum genome via pfam, KEGG, and GO analyses. The 63 genes that were induced in common between PyOM and burned soil, excluding water, characterize a common response to pyrolyzed substrates . In addition, 335 genes were uniquely upregulated in response to PyOM, and the 38 genes uniquely upregulated on burned soil were almost entirely annotated as hypothetical proteins . After “hypothetical,” the next category with the most genes was that of metabolism, which we address in the subsequent section. We note that PyOM-responsive genes included nine Cytochrome P450 monooxygenases and four FAD monooxygenases. Cytochrome P450 oxidation of aromatic compounds often results in the formation of toxic epoxides and reactive oxygen species . On both substrates containing PyOM, we observed upregulation of genes involved in ROS protection . However, neither of the two epoxide hydrolases annotated in the P. domesticum genome exhibited any significant changes across our treatments . Lastly, we observed an enrichment of genes involved in biosynthesis , development, and signaling that were upregulated specifically in the presence of PyOM. Taken together, these data indicate that, as expected, growth on PyOM is more stressful than growth on sucrose. Beyond a general stress response,dutch buckets the P. domesticum response to burned or pyrolyzed substrates includes the activation of a large set of genes, including those involved in metabolism, oxidation of aromatic substrates, and protection from ROS.

The results in the previous section indicate that PyOM may prompt a restructuring of metabolism in P. domesticum. In Figure 4, we mapped the significantly upregulated genes in P. domesticum onto the canonical pathways for aromatic compound degradation and assimilation into central metabolism and other biosynthetic pathways in fungi . All PyOM is enriched for aromatic carbon compounds because incomplete combustion of organic matter results in the formation of aromatic and polyaromatic carbon compounds . PyOM produced at temperatures greater than ~400°C generally has a carbon composition that is >90% aromatic, and as temperature increases, the size of polyaromatic compounds also increases . Here, we propose that the large cohort of cytochrome P450 and FAD monooxygenases that were induced on PyOM-containing media are the primary method that P. domesticum uses to initiate the degradation of polyaromatic and aromatic carbon compounds. FAD monooxygenases oxidize compounds with a single aromatic ring, whereas cytochrome P450 monooxygenases can oxidize complex polyaromatic compounds . One cytochrome P450 gene that was upregulated on both PyOM and burned soil was identified via proteinBLAST as a putative ortholog of the bapA gene in A. nidulans, which was recently shown to oxidize the polyaromatic hydrocarbon benzo-[a]-pyrene . Notably, the conserved C-terminus of the BapA protein is largely conserved in P. domesticum, including conserved signature motifs . An additional five upregulated FAD monooxygenase genes and one cytochrome P450 monooxygenase gene were identified during genome annotation to have homology with proteins that have specific predicted substrates .

Lastly, 15 cytochrome P450 monooxygenase genes were induced at least 2-fold on PyOM-containing media that have homology to monooxygenase proteins with currently unknown substrates . Nearly half of these genes were strongly induced on PyOM; gene_10112, encoding a cytochrome P450 was strongly upregulated on both PyOM compared to sucrose and on PyOM compared to water , and six other cytochrome P450 genes were also upregulated at least 8-fold on PyOM compared to sucrose. Assimilation of aromatic carbon into central metabolism occurs primarily via two pathways in fungi: either via protocatechuate and the shikimate/quinate pathway , or via catechol and the 3-oxoadipate pathway . Six of seven core genes in the shikimate/quinate pathway were upregulated on PyOM compared to sucrose. In contrast, two of the five genes in the 3-oxoadipate pathway were upregulated on PyOM compared to sucrose. Notably, we observed strong upregulation on PyOM compared to sucrose of the four genes necessary to connect aromatic protocatechuate to central metabolism. These four genes encode DHS dehydrase , DHQase , DHQ synthase , and DAHP synthase . These three genes were similarly strongly upregulated on PyOM compared to water . In contrast, the genes that encode the proteins necessary for the 3-oxoadipate pathway were relatively modestly upregulated on PyOM compared to sucrose . We also observed that genes required for the breakdown and metabolism of the three aromatic amino acids was induced differentially across all tested conditions. It is notable that upregulation of monophenol monooxygenase genes was also enriched on burned or pyrolyzed substrates and water compared to sucrose. Upregulation of central metabolism genes was generally enriched on sucrose; however, some genes involved in glycolysis and gluconeogenesis were also upregulated on water, PyOM, and soil. In summary, when P. domesticum was grown on PyOM, we observed upregulation of an extensive set of monooxygenases that may initiate degradation of the aromatic components of PyOM.

We also observed comprehensive induction of the shikimate/quinate and 3-oxoadipate pathways, though the shikimate/quinate pathway was much more strongly induced. These data indicate that the aromatic intermediates liberated by monooxygenases may be funneled into central metabolism and mineralized via the shikimate/quinate and 3-oxoadipate pathways in P. domesticum.Fungi in the genus Pyronema are pioneer species that rapidly dominate fungal communities in post-fire soils . Thus, Pyronema have the potential to directly influence the trajectory of post-fire community succession and associated nutrient cycling dynamics. Here, we investigated the transcriptional response of P. domesticum on four different agar treatments: 750°C PyOM, wildfire burned soil, sucrose minimal medium, and water. Our results indicate that burned or pyrolyzed substrates induce transcription of a comprehensive set of genes that together function to metabolize aromatic and polyaromatic compounds found in PyOM. Additionally, we demonstrated the mineralization of PyOM into CO2 by P. domesticum, consistent with the notion that this organism is capable of directly metabolizing high-temperature PyOM. Pyronema are barely detectable in soil before fire, become prevalent soon after fire, and then rapidly decline within weeks . The form taken by Pyronema between fire events is largely obscure. Pyronema may simply exist as dormant ascospores or sclerotia that require the heat and/or chemical changes associated with fire to break dormancy and initiate germination . For example, plant-derived polysaccharides such as hemicellulose burned at high temperatures result in the formation of furfural, which triggers germination of N. crassa ascospores . One recent hypothesis suggests that pyrophilous fungi may live as endophytes for the majority of their life history, abandoning their plant hosts after they are killed by fire . Regardless of how Pyronema live pre-fire, post-fire Pyronema are clearly poised to take full advantage of an open niche. Past work has shown that Pyronema are poor competitors, and they are also capable of growing rapidly on a diversity of substrates . These data point toward the notion that Pyronema are generalists. During intense forest fires, the organic material in the topmost layer of soil is heavily pyrolyzed, ultimately containing a significant amount of PyOM, with patches where the soil surface is covered in a layer of pure PyOM . PyOM is a heterogeneous material composed of complex aromatic and polyaromatic carbon compounds . Beyond PyOM,grow bucket this top layer of soil is depleted of easily-metabolized C sources and other macronutrients . A secondary layer of soil beneath the top layer is heated to a point that causes widespread death of the resident microbial/invertebrate soil fauna, leading to a layer rich in necromass that is not pyrolyzed . Carbon found in either layer could be targeted by Pyronema. The notion of Pyronema as generalists might suggest that they would be most likely to exploit the readily available carbon in the necromass layer. In contrast, growth on PyOM-containing agar, the metabolic restructuring at the transcriptional level, and production of 13C-labeled CO2 from labeled 750°C PyOM that we observed in this study indicate that P. domesticum is capable of metabolizing PyOM. Specifically, this metabolic restructuring includes the activation of an array of cytochromes P450 and FAD monooxygenases which may target aromatic substrates for oxidation. Additionally, we observed robust activation of the shikimate/quinate and 3-oxoadipate pathways for assimilating the resulting substrates into central metabolic pathways . Notably, we did not observe any significant differential expression of epoxide hydrolase genes in P. domesticum, indicating that pathway may not be relevant in P. domesticum. Together our results indicate that Pyronema may in fact be well adapted as broad generalists able to capitalize on both necromass and abundant PyOM in post-fire soils, including high-temperature PyOM, which is often assumed to be recalcitrant and lacking in bioavailable carbon .

Furthermore, this broad generalist behavior may explain how Pyronema are able to rapidly dominate postfire soil communities. Although their prevalence is relatively short-lived, Pyronema grow rapidly post-fire, producing abundant biomass in the form of ascocarps and mycelia . However, both Pyronema ascocarps and their DNA decline rapidly after they peak in abundance following fire . Some decline of Pyronema DNA could be explained by the starvation response that we observed on water agar , in which P. domesticum may fuel outward expansion by recycling macromolecular building blocks such as nucleotides and amino acids into a diffuse biomass aimed at exploration of environments with sparse nutrients . This turn-over of biomass may account for some of the non-PyOM-derived CO2 detected in our experiments . Alternatively, P. domesticum may have simply mineralized other carbon sources that were present in the agar medium, such as impurities in the agar itself. To our knowledge, terrestrial fungi lack agarases that degrade agarose, but the genomes of fungi such as P. domesticum do contain a suite of pectinases, some of which may target agaropectin . The data in Figure 5B demonstrate that P. domesticum is capable of mineralizing carbon from PyOM produced at 750°C, which yields material that is completely pyrolyzed, and hence challenging to break down biologically. Furthermore, the finding that P. domesticum produced CO2 from scarce, non-PyOM sources inthe presence of abundant PyOM implies that they likely prefer to metabolize other carbon substrates over PyOM if they are available. While such a metabolic hierarchy makes sense from an energetic standpoint, it also reflects the ecology of these organisms. For example, Pyronema may grow abundantly on C available in the necromass layer and only switch to metabolizing PyOM after those resources are exhausted or during surface growth during fruiting body formation. The short-lived dominance of Pyronema might also be explained by competition, as it appears to be a weak competitor in isolation . However, even if Pyronema are outcompeted and simply senesce, their DNA could linger in post-fire soil and continue to be detected via sequencing methods . Thus, another possible explanation for the rapid decline of Pyronema DNA in post-fire soils is that Pyronema biomass, either living or recently senesced, is consumed by other organisms . Thus, abundant Pyronema biomass may provide a critical nutrient source for secondary colonizers of post-fire soils, thereby laying the foundation for succession within post-fire communities. Importantly, the ability of P. domesticum to convert some PyOM into biomass could directly facilitate the growth of organisms that lack the ability to metabolize PyOM. Thus, Pyronema may provide an important mechanism for rapidly assimilating some portion of newly formed PyOM back into more readily bioavailable forms of carbon in post-fire environments. Additionally, it is possible that Pyronema function to favorably transform the post-fire soil environment in other ways, such as affecting pH or accessibility of other nutrients. Nevertheless, the mineralization of PyOM by the dominant early-successional fungus P. domesticum is likely to have broad impacts on post-fire succession and recovery in soil microbial communities.Chardonnay and Pinot noir are two premium wine grape varieties cultivated in California. In 2020, Chardonnay continued to be the leading white wine grape variety in California at 539,321 tons , while Pinot noir was at 212,590 tons for red wine grape variety . According to the latest California Agricultural Production Statistics, grapes ranked third among California’s top-10 valued commodities for the 2020 crop year generating $4.48 billion in cash receipts. Behind these impressive figures, the California wine industry currently suffers from a relatively high degree of waste as roughly 20% of the grape mass goes unused after wine making . There are two main grape by-products generated in the wine industry.