Chemical and Biological Diversity of the Xylariales, with emphasis on Polythetic Phylogenomic Studies of the Hypoxylaceae

Chemical and Biological Diversity of the Xylariales, with emphasis on Polythetic Phylogenomic Studies of the Hypoxylaceae Virtual Lecture, International Conference on Exploring Fungal Frontiers: A Multidisciplinary Approach to Advancing Fungal Biology & 3rd Annual Meet of Association of Fungal Biologists (AFB) Kolhapur, 5 Dec. 2025 Marc Stadler, Dept. Microbial Drugs, HZI Braunschweig, Germany; [email protected] Group photo of Department MWIS, June 2023 Ca. 60 people from > 15 countries Name der Präsentation Seite 3 | 4 Strong competition in tropical rainforests Image by Thomas Læssøe (Khao Yai NP, Thailand, 2007) Amauroderma sp. (Basidiomycota, Ganodermataceae) Squamotubera leratii (Ascomycota, Xylariaceae) Xylariales – Stromata & sexual structures Images taken from Stadler M, et al. (2013) Mycology 4: 5-21. Seite 6 | Over 4.000 accepted species, and many more remain to be discovered Highest diversity in the tropics Cosmopolitan, ubiquitous wood-degraders Predominant fungal endophytes of most seed plants Associated with insect vectors Some species are important plant pathogens Model example for the study of correlations between chemical and biological diversity! Stadler M (2011). Curr Res Environm Appl Mycol 1:75-133. Seite 7 | Waxy tissue,with orange-brown granules, containing strongly bioactive daldinins Perithecia “woody” stromal tissue Substrate (bark of Corylus) Large amounts of bioactive pigments (up to 20% of dry weight!!) beneath stromatal surface Image by Jacques Fournier Section through a stroma of Hypoxylon fuscum O CH3 CH3 O O CH3 AcO O O CH3 OH O Daldinin C Stadler M, Fournier J (2006) Rev. Iberoam. Micol. 23: 160-170 Stromatal anatomy and secondary metabolites of Hypoxylon Purple stromal surface (containing BNT) OH O H OH O H Seite 8 | ! Stadler M et al. (2008) North American Fungi 3: 73-125 HPLC profile of type specimen, collected 1796 Ancient herbarium specimens have usually retained their characteristic metabolite patterns over the centuries 200.00 300.00 400.00 500.00 600.00 0 100 % Combine (105:107-(87:88+141:142)) 381.23 149.11167.12 321.30 309.20 441.26 385.30 459.33 489.28 0 100 % Combine (105:107-(87:88+141:142)) 443.32 293.28 387.37 444.23 -158 100 % Combine (1686) 224.00 332.00 268.00 nm250 300 350 400 450 500 550 mAU 0 50 100 150 200 250 O O OO O OH OH O O Rubiginosin A Species specific HPLC fingerprints Archaeological specimens associated with charcoal Found during excavations in the Châtillon-sur-Seine forest massif, Central France by a group of archaeologistsfrom the Sorbonne university, Paris Charcoal and soil were dated by radiocarbon and thermoluminescence to the medieval period (between 738 and 1411 AD) Reminiscent of Hypoxylaceae stromata Meticulous and tedious microsopy to search for ascospores and HPLC profiling of the stromatal extracts Frank Surup et al., (2018) Fungal Divers 92:345-356. Xylarialean asexual states Predominant fungal endophytes (thousands of orphan ITS DNA sequences in GenBank) Stadler M et al. (2013). Mycology, 4: 5-21. Families re-organised according to anamorph types! Libertella-like (Diatrypaceae, Lopadostomataceae) Geniculosporium-like (Xylariaceae sensu stricto) Nodulisporium-like (Hypoxylaceae, Graphostromataceae) Xylocladium state (Camillea, Biscogniauxia) Nodulisporium state Wendt et al. (2018) Mycol Prog.; Daranagama et al. (2018) Fungal Divers. Potent insecticide from xylariaceous endophytes www.eol.org Nodulisporic acid A Source: Bontia daphnoides, collected in Hawaii, USA Nodulisporium type endophyte Strain MF5954 (Merck Collection) Developmental product as antiparasitic agent (Merck) Bills GF et al. (2012), PLoS ONE 7(10): e46687. Corresponding teleomorph: Hypoxylon pulicicidum Bills GF et al. (2012), PLoS ONE 7(10): e46687. Nodulisporic acid A Found in the Caribbean (Martinique) many years after the discovery of the first endophytic producer strains Biogeography of Hypoxylon pulicicidum AA New „virtual collection sites“ Bills GF et al. (2012), PLoS ONE 7(10): e46687. Results of a BLAST search in GenBank after having assured specificity of ITS sequence Collection sites of endophytes identified by Merck researchers Holotype location Collection site of original producer PF-1022A and emodepside N N N N O O O O O O O OO O O O H H H H H H H H N N O O PF-1022A - Natural product from an endophytic xylariaceous fungus Emodepside - Semisynthetic PF1022A derivative, showing higher efficacy in vivo than its parent compound Emodepside, a product of Bayer (Animal Health) as antiparasitic agent, Up to date the only marketed drug derived from an endophytic organism ! Field work in and taxonomic expertise from Argentina Esteban B. Sir (Tucuman, Argentina) Characteristic morphological features Geniculosporium (true Rosellinia) Dematophora Pathogenic Rosellinia are transferred into Dematophora Hartig 1865 (resurrected genus) K. Wittstein et al. (2020) Stud Mycol 96:1-16. Dematophora asexual state (contains all known pathogens) Geniculosporium asexual state (contains no dangerous pathogens!) Kathrin Wittstein Pathogenic Rosellinia s. lat can be moved into Dematophora Hartig! New terpenoids but no PF-1022A found from Dematophora bunodes and D. pepo PF-1022A obtained from ascospore isolates of Rosellinia corticium for the first time (identity checked by HRMS and NMR!) Christopher Lambert K. Wittstein et al. (2020) Stud Mycol 96:1-16. Synteny analysis reveals similar BGC in the genomes Detection of homologous BGC even in species that do not apparently produce the respective metabolites as shown by HPLC analytics Different types of a certain BGC class (e.g. azaphilones) are easily discernible Kuhnert et al. 2021, Stud.. Mycol Elucidation of the azaphilone biosynthesis in H. fragiforme Identification of the hybridorubrin/fragiformin BGC (C) was accomplished by genome analysis/homology search Known BGC encoding for mitorubrins (A, B) were found as best matches K. Becker et al., (2021) Chemistry Eur J . Zeng et al. JOF 9:726 (2023) Haoxuan Zeng New genus justified by chemotaxonomic evidence & molecular phylogeny Christopher Lambert Sporothriolides = Genus-specific marker metabolites with selective antifungal effects Species-specific metabolites of Hypoxylon monticulosum Seite 36 | O O O O H HO O O O H HHO O O O H H HO OOH O O H OH OOH O O H OH 123 4 5 Chemical structures of sporothriolide (1), dihydrosporothriolide (2), sporothric acid (3), isosporothric acid (4) and dihydroisosporothric acid (5) from cultures of Hypoxylon monticulosum. Surup et al. (2014) Mycol Int J Fungal Biol 5: 110-119. Major metabolite for H. monticulosa (cultures from Africa Asia & America) Apparently selective antifungal activity, not cytotoxic and antibacterial Not found in >200 cultures of other Hypoxylon spp. Species-specific metabolites of Hypomontagnella monticulosa Preliminary data: Sporothriolide is a selective antifungal agent! Surprisingly, no cytotoxicity and no antibacterial effects were observed, despite the presence of an exo-methylene keto moiety ! Biosynthesis of sporothriolides and sporochartins Several key enzymes identified by heterologous expression Tian et al. (2020) Chem. Sci. 11:12477-12484 Alkyl citrates and their biosynthesis Feeding experiments Tian et al. (2020) Chem. Sci. 11:12477-12484 Transcriptome studies (and concurrent HPLC analysis) Stromatal development in H. fragiforme &H. howeanum Conidial stage of H. howeanum Immature stromata April White granules below the stromatal surface April Mature stromata July White granules have disappeared M. Stadler et al.: Myc. Res. 110, 811-820 (2006) Over 150 different metabolites were detected by HPLC-DAD/MS in different developmental stages of a single species The highest metabolic diversity was observed in the maturing stromata and the anamorph colonising the natural substrates Follow up on this study at HZI has resulted in several intersting results! Maturing stromata: Hotspots of metabolic diversity! O RO O O O HO HO N HO OOH OH OHO OH OH Unknown Known min 0 2 4 6 8 10 12 14 Norm. 0 100 200 300 400 500 600 DAD1 C, Sig=210,8 Ref=550,100 (XYLAR\14100501.D) 0.875 1.285 2.187 3.099 3.400 3.936 4.158 4.530 4.741 4.899 5.138 5.362 5.692 5.854 6.001 6.185 6.798 7.209 7.406 7.631 7.779 8.163 8.426 8.697 9.050 9.245 9.657 10.007 10.224 10.399 10.607 10.790 11.022 11.263 11.442 11.711 11.943 12.171 12.567 12.712 13.671 M. Stadler et al.: Myc. Res. 110, 811-820 (2006) Cytochalasins! SAR of cytochalasans on actin cytoskeleton Compound activity Reversible Cytochalasin B ++ + Cytochalasin D +++ +/- Cytochalasin F + + Cytochalasin H +++ + Cytochalasin Z2 + + Cytochalasin 6 ++ - Cytochalasin 9 + +/- Cytochalasin 10 + +/- Cytochalasin 11 - + Cytochalasin 12 + +/- 18-epi-cytochalasin 12 +++ - Epoxycytochalasin C +++ + Epoxycytochalasin D +++ +/- Epoxycytochalasin N + + Epoxycytochalasin Q ++ + L-696,474 +++ + 21-O-deacyl-L696,474 +++ + Deoxaphomin +++ - 18-deoxy-fragiformin A + +/- 18-epi-fragiformin B +++ - Cytochalasin 116 - + Tyrosine-cytochalasin 1 ++ - Tyrosine-cytochalasin 2 - + Chaetoglobosin A + + Chaetoglobosin D ++ - Sorbicillin +++ + Ratings of cytochalasin related effects on U2OS cells: +++ = actin aggregation and cell shape disruption at 1 µg/ml; ++ = slight actin aggregations at 1 µg/ml visible, no disruption of cell shape; + = aggregation or cell shape disruption visible at 5 µg/ml; - = no cytochalasin related effects observable. Ratings of reversibility of cytochalasin related effects: + = reversible, no cytochalasin related effect observable after wash out; +/- = partially irreversible, actin aggregation and cell shape disruption not as severe as 5 µg/ml concentration of compound; - = irreversible, cytochalasin related effect after wash out similar to the effect at a compound concentration of 5 µg/ml. Differential activities Weak cytotox, reversible Potential antivirals (virus entry inhibitors) Strong cytotox, irreversible: Anticancer compounds (even usable as conjugates) R. Kretz et al. (2019) Biomolecules 9:73 C. Wang et al. (2019) Org. Lett Optimisation of cytochalasans using methods of synthetic biotechnology Modified cytochalasins as tool compounds Mutasynthesis => Linking to various dyes C. Wang et al. (2020) Chemistry Eur J, 26: 13578-13583 Modified cytochalasins as tool compounds e.g. for visualization of intracellular actin structures C. Wang et al. (2020) Chemistry Eur J, 26: 13578-13583 Chemotaxonomic segregation criteria Jackrogersella (= „A. multiforme“ complex; papillate ostioles) contains cohaerin/multiformin type azaphilones as stromatal pigments Annulohypoxylon spp. (ostiolar rings) are devoid of these azaphilones and predominantly contain binaphthalene derivatives instead Typical stromatal metabolites of: Annulohypoxylon s. str. Jackrogersella gen. nov. Common metabolite Jack D. Rogers † Detection of cohaerin-like azaphilone BGC Kuhnert et al. 2021, Stud.. Mycol New genus segregated from Hypoxylon Basal clade in Hypoxylon: New genus Parahypoxylon Cedeño-Sanchez,Charria-Girón, Lambert et al., MycoKeys (2022) Detection of cohaerin-like azaphilone BGC Kuhnert et al. 2021, Stud.. Mycol Genus-specific metabolite profiles HPLC profiles remain stable for centuries even here! Type material from 1893 vs. fresh specimens collected in 2007 Base peak chromatograms (BPCs) from UHPLC-MS analysis of stromatal extracts Common peaks in red shade Cedeño-Sanchez,Charria-Girón, Lambert et al., MycoKeys (2022) Marjorie Cedeño Esteban Charria Antiviral azaphilones: Cohaerins Jansen-Ohliges et al. 2022, Cells 12:83 Cohaerin-type azaphilones: Specific for Jackrogersella spp. and some Hypoxylon spp. Lili Jia Name der PräsentationSeite 64 | Molecular phylogenetic maximum Likelihood (lLn= -148775.562) tree inferred from multigene alignment (whole ACT1, TUB2, TEF and RPB2). TEF was never before used in a phylogeny of the Xylariales ! Housekeeping genes can be retrieved from the genomes without problems (no primers necessary!) M. Cedeño-Sanchez, Tian Cheng, C Lambert et al., unpublished Outlook Seite 65 | Phylogenomics based on the >60 3rd generation genome sequences of Hypoxylaceae Final goal: New world monograph of the genus Hypoxylon based on polythetic taxonomy and phylogenomics Increase the data matrix of our metabolomics platform for dereplication based on HPLC-MS/MS techniques to support the search for new compounds in the stromata and cultures of these fungi The biosynthesis of further compound classes is being elucidated based on methods of synthetic biotechnology (collaboration with the Cox and Collemare groups) Acknowledgements In-house collaboration at HZI/HIPS: Theresia Stradal, Mathias Müsken, Mark Brönstrup, Rolf Müller & coworkers Co-authors of papers on Thai fungi; above all Jennifer Luangsa-ard, Kevin D. Hyde & co-workers Pedro W. Crous, Jerome Collemare (WFBI), Miroslav Kolarik (IMIC Prague) and co-workers Collaborators in the ERAFRICA project: Josphat Matasyoh (Kenya), Cony Decock (MUCL Belgium), TaxonOmics project: Russell Cox, Eric Kuhnert, (LU Hannover), Jörn Kalinowski (CEBITEC Bielefeld) and coworkers AvH postdocs: Remy Teponno (Cameroon), Mark Kimani (Kenya) Soleiman Helaly † & Sherif Ebada (Egypt) Wolfgang Maier (JKI Braunschweig) and co-workers; Expert technical assistance: Wera Collisi, Anke Skiba, Christel Kakoschke, Silke Reinecke, Kerstin Schober & Aileen Gollasch (HZI) and many others Various funding agencies Africa trip June 2025 Mycobiomics EU-H2020-MSCA-RISE project •This research benefitted from funding by the European Union’s Horizon 2020 research and innovation program (RISE) under the Marie Skłodowska-Curie grant agreement No. 101008129, project acronym “Mycobiomics”.