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Cyclocybe parasitica is a wood-destroying parasitic edible mushroom growing on diverse broad-leafed trees in New Zealand and other Pacific areas. Recent molecular systematics of European \(\textit {Cyclocybe aegerita}\), a newly delimited Asian phylum and of related species, corroborated the distinction of the chiefly saprobic cultivated edible mushroom \(\textit {C. aegerita}\) from \(\textit {C. parasitica}\). Here, we show that \(\textit {C. parasitica}\) exhibits a morpho-physiological trait characteristic to its European cousin, i.e., monokaryotic fruiting \(\textit {sensu stricto}\) (basidiome formation without mating). Monokaryotic fruiting structures formed by \(\textit {C. parasitica}\) ICMP 11668-derived monokaryons were categorized into four phenotypes. One of them displays ulcer-like structures previously reported from bracket fungi. Histology of dikaryotic and monokaryotic \(\textit {C. parasitica}\) fruiting structures revealed anatomical commonalities and differences between them, and towards monokaryotic fruiting structures of \(\textit {C. aegerita}\). Mating experiments with \(\textit {C. parasitica}\) strains representative of each fruiting phenotype identified compatible sibling monokaryons. Given reports on hypothetically monokaryotic basidiome field populations of \(\textit {"C. aegerita sensu lato"}\), it seems worthwhile to prospectively investigate whether monokaryotic fruiting s.str. occurs in nature. Sampling from such populations including karyotyping, comparative -omics, and competition assays may help to answer this question and provide evidence whether this trait may confer competitive advantages to a species capable of it.

Additional file 7: Revealing putative enzymes of the oxylipin pathway in C. aegerita. Figure S12. Phylogenetic analysis of different DOXs. Figure S13. Partial amino acid sequence alignment of different DOXs. Figure S14. Phylogenetic analysis of different CYP74 proteins. Figure S15. Transcription levels of the putative DOX AAE3_13098 and the putative HPL AAE3_09203. Figure S16. Transcription levels of AAE3_04864 (LOX4) and the putative HPL AAE3_05330. Figure S17. Phylogenetic analysis of putative ADHs. Figure S18. Amino acid sequence alignment of ene-reductases. Figure S19. Transcription levels of the putative ADHs AAE3_00054 and AAE3_06559 as well as the putative ene-reductase AAE3_13549.

Additional file 6: Figure S8. Proposed pathways for the enzymatic formation of fungal oxylipins. Figure S9. Transcription levels of putative HPLs as well as the relative concentrations of volatile oxylipins in the headspace of C. aegerita. Figure S10. Transcription levels of putative ADHs as well as the relative concentrations of volatile oxylipins in the headspace of C. aegerita. Figure S11. Transcription levels of putative ene-reductases as well as the relative concentrations of volatile oxylipins in the headspace of C. aegerita.

Additional file 3: Transcription of fruiting-related genes (FRGs). Figure S2. Correlation of the expression of putative C. aegerita homologs of FRGs in plectenchymatic samples (FB ‘tissue’) during the fructification process. Figure S3. Correlation of the expression of putative C. aegerita homologs of FRGs in mycelium samples. Figure S4. RT-qPCR-based expression level assessment with C. aegerita orthologs of four well-known fruiting-related genes (FRGs) during fruiting of C. aegerita. Table S4: RT-qPCR primers for the FRGs HOM1, GAT1, BWC2 and DST1.

Additional file 4: Terpenoid biosynthesis. Figure S5. Expression of genes involved in the mevalonate pathway and of the sesquiterpene synthases Agr1 to Agr9 in C. aegerita. Figure S6. Transcription levels of the sesquiterpene synthases Agr1, Agr4, Agr8 and Agr9.

Additional file 1: Differential gene expression during fruiting body development. Figure S1. Heatmap of DEGs in mycelium and FB samples.

Additional file 5: The fatty acid metabolism. Figure S7. Expression of genes putatively involved in the fatty acid biosynthesis of C. aegerita.

Fruiting body-forming members of the Basidiomycota maintain their ecological fitness against various antagonists like ascomycetous mycoparasites. To achieve that, they produce myriads of bioactive compounds, some of which are now being used as agrochemicals or pharmaceutical lead structures. Here, we screened ethyl acetate crude extracts from cultures of thirty-five mushroom species for antifungal bioactivity, for their effect on the ascomycete Saccharomyces cerevisiae and the basidiomycete Ustilago maydis. One extract that inhibited the growth of S. cerevisiae much stronger than that of U. maydis was further analyzed. For bioactive compound identification, we performed bioactivity-guided HPLC/MS fractionation. Fractions showing inhibition against S. cerevisiae but reduced activity against U. maydis were further analyzed. NMR-based structure elucidation from one such fraction revealed the polyyne we named feldin, which displays prominent antifungal bioactivity. Future studies with additional mushroom-derived eukaryotic toxic compounds or antifungals will show whether U. maydis could be used as a suitable host to shortcut an otherwise laborious production of such mushroom compounds, as could recently be shown for heterologous sesquiterpene production in U. maydis.

Fruiting body-forming members of the Basidiomycota maintain their ecological fitness against various antagonists like ascomycetous mycoparasites. To achieve that, they produce myriads of bioactive compounds, some of which are now being used as agrochemicals or pharmaceutical lead structures. Here, we screened ethyl acetate crude extracts from cultures of thirty-five mushroom species for antifungal bioactivity, for their effect on the ascomycete \(\textit {Saccharomyces cerevisiae}\) and the basidiomycete \(\textit {Ustilago maydis}\). One extract that inhibited the growth of \(\textit {S. cerevisiae}\) much stronger than that of \(\textit {U. maydis}\) was further analyzed. For bioactive compound identification, we performed bioactivity-guided HPLC/MS fractionation. Fractions showing inhibition against \(\textit {S. cerevisiae}\) but reduced activity against \(\textit {U. maydis}\) were further analyzed. NMR-based structure elucidation from one such fraction revealed the polyyne we named feldin, which displays prominent antifungal bioactivity. Future studies with additional mushroom-derived eukaryotic toxic compounds or antifungals will show whether \(\textit {U. maydis}\) could be used as a suitable host to shortcut an otherwise laborious production of such mushroom compounds, as could recently be shown for heterologous sesquiterpene production in \(\textit {U. maydis}\).

\(\textit {Cyclocybe aegerita}\) (synonym: \(\textit {Agrocybe aegerita}\)) is a widely cultivated edible and reportedly almost cosmopolitan mushroom species that serves as a model fungus for basidiome formation and as producer of useful natural products and enzymes. Focusing on strains from different continents, here, we present a phylogenetic analysis of this species and some adjacent taxa that employs four phylogenetic markers. In addition, we tested the strains' capability to fructify on agar media. Our analysis reveals that "\(\textit {C. aegerita sensu lato}\)" splits up into the following two well-supported monophyletic geographic lineages: a European clade and an Asian clade. The European one is closely associated with the Chinese species \(\textit {Cyclocybe salicaceicola}\). In contrast, the Asian lineage, which we preliminarily designate as \(\textit {Cyclocybe chaxingu}\) agg., may comprise several species (species complex) and clusters with the Pacific species \(\textit {Cyclocybe parasitica}\) (New Zealand). In addition, fruiting properties differ across \(\textit {C. aegerita}\) and its Asian and Pacific relatives; however, strains from the Asian clade and \(\textit {C. parasitica}\) tend to form larger basidiomes with relatively big caps and long stipes and strains from the European clade exhibit a more variable fruiting productivity with the tendency to form more basidiomes, with smaller caps and shorter stipes. Moreover, some strains showed individual fruiting patterns, such as the preference to fruit where they were exposed to injuring stimuli. In conclusion, the delimitation of the newly delimited Asian species complex from our multilocus phylogeny of "\(\textit {C. aegerita sensu lato}\)", which is supported by phenotypic data, depicts an exemplary case of biogeographic diversity within a previously thought homogeneous species of near worldwide distribution.

The strophariaceous basidiomycete Cyclocybe aegerita (synonyms Agrocybe aegerita and A. cylindracea) is one of the most praised cultivated edible mushrooms and is being cultivated at large scale for food production. Furthermore, the fungus serves as a model organism to study fruiting body formation and the production of secondary metabolites during the life cycle of Basidiomycota. By studying the secondary metabolite profiles of C. aegerita, we found several terpenoids in submerged cultures. Aside from the main metabolite, bovistol (1), two new bovistol derivatives B and C (2, 3) and pasteurestin C as a new protoilludane (4) were isolated by preparative HPLC. Their structures were elucidated by mass spectrometry and NMR spectroscopy. The relative configurations of 2-4 were assigned by ROESY correlations, and 3JH,H coupling constants in the case of 4. Applying quantitative PCR for gene expression validation, we linked the production of bovistol and its derivatives to the respective biosynthesis gene clusters.

Agrocybe aegerita AAE-3 genes in subcategories of GO term â cellular componentsâ . (DOCX 31 kb)

Agrocybe aegerita AAE-3 genes in subcategories of GO term â molecular functionâ . (DOCX 31 kb)

Agrocybe aegerita AAE-3 genes in subcategories of GO term â biological processesâ . (DOCX 31 kb)

Proteins encoded by fruiting-related genes from different agaricomycetes species. (DOCX 34 kb)

Supplementary Figures. (PDF 1068 kb)

Annotation of Agrocybe aegerita AAE-3 genes using InterProScan. (DOCX 254 kb)