This study investigated the composition and temporal dynamics of wood-inhabiting fungal communities in four aging tree species in Lednice Castle Park (Czech Republic), located within the Lednice-Valtice Cultural Landscape, a UNESCO World Heritage Site. Forty wood cores were collected from 20 trees at two time points (2023 and 2024). The hosts included horse chestnut (Aesculus hippocastanum L.), copper beech (Fagus sylvatica 'Atropunicea' L.), oak (Quercus robur L.), and poplar (Populus alba L.), each exhibiting visual signs of decline. Fungal assemblages were profiled using ITS2 high-throughput amplicon sequencing. Ascomycota dominated across all hosts (72-89% of reads), while Basidiomycota contributed 8-24%, largely represented by Agaricomycetes in F. sylvatica. Alpha diversity varied significantly among hosts (Shannon: F3,36 = 10.61, p = 0.001 in 2023; F3,36 = 10.00, p = 0.001 in 2024). Temporal shifts were host-dependent: F. sylvatica exhibited the strongest year-to-year decline in richness (Chao1: -83%, p = 0.007) and increased beta dispersion, while A. hippocastanum and P. alba showed significant increases in diversity (+65% and +42%, respectively). Community composition was shaped by host species (PERMANOVA Bray-Curtis: p = 0.001) and shifted over time (Jaccard: p = 0.001), with F. sylvatica showing the highest temporal turnover. Functional guild analysis revealed consistent dominance of saprotrophs (29-41%) and mixed pathotroph-saprotroph guilds (23-36%) across hosts, indicating active degradation processes inside functional xylem. These results indicate that, within the studied system, the wood mycobiome of aging trees is host-dependent and temporally dynamic rather than static or functionally neutral. Short-term temporal turnover observed between sampling years may contribute to shifts in fungal community composition and succession within wood, with potential implications for tree decline processes in managed historical park landscapes.

Department of Planting Design and Maintenance Mendel University in Brno Valticka 334 691 44 Lednice Czech Republic

Mendeleum Institute of Genetics Mendel University in Brno Valticka 334 691 44 Lednice Czech Republic

Zobrazit více v PubMed

Blicharska M., Mikusinski G. Incorporating Social and Cultural Significance of Large Old Trees in Conservation Policy. Conserv. Biol. 2014;28:1558–1567. doi: 10.1111/cobi.12341. PubMed DOI

Konijnendijk C. The Forest and the City-the Cultural Landscape of Urban Woodland. Springer; Berlin/Heidelberg, Germany: 2008.

Stagoll K., Lindenmayer D.B., Knight E., Fischer J., Manning A.D. Large trees are keystone structures in urban parks. Conserv. Lett. 2012;5:115–122. doi: 10.1111/j.1755-263X.2011.00216.x. DOI

Lindenmayer D.B., Laurance W.F., Franklin J.F. Global Decline in Large Old Trees. Science. 2012;338:1305–1306. doi: 10.1126/science.1231070. PubMed DOI

Fay N. Environmental arboriculture, tree ecology and veteran tree management. Arboric. J. 2002;26:213–238. doi: 10.1080/03071375.2002.9747336. DOI

Schwarze F.W.M.R., Engels J., Mattheck C. Fungal Strategies of Wood Decay in Trees. Springer; Berlin/Heidelberg, Germany: 2000.

Lonsdale D. Principles of Tree Hazard Assessment and Management. Stationery Office; London, UK: 1999.

Parfitt D., Hunt J., Dockrell D., Rogers H.J., Boddy L. Do all trees carry the seeds of their own destruction? PCR reveals numerous wood decay fungi latently present in sapwood of a wide range of angiosperm trees. Fungal Ecol. 2010;3:338–346. doi: 10.1016/j.funeco.2010.02.001. DOI

Boddy L., Heilmann-Clausen J. Chapter 12 Basidiomycete community development in temperate angiosperm wood. In: Boddy L., Frankland J.C., van West P., editors. British Mycological Society Symposia Series. Volume 28. Academic Press; Cambridge, MA, USA: 2008. pp. 211–237.

Gilmartin E.C., Jusino M.A., Pyne E.J., Banik M.T., Lindner D.L., Boddy L. Fungal endophytes and origins of decay in beech (Fagus sylvatica) sapwood. Fungal Ecol. 2022;59:101161. doi: 10.1016/j.funeco.2022.101161. DOI

Martin R., Gazis R., Skaltsas D., Chaverri P., Hibbett D. Unexpected diversity of basidiomycetous endophytes in sapwood and leaves of Hevea. Mycologia. 2015;107:284–297. doi: 10.3852/14-206. PubMed DOI

Fukasawa Y. Ecological impacts of fungal wood decay types: A review of current knowledge and future research directions. Ecol. Res. 2021;36:910–931. doi: 10.1111/1440-1703.12260. DOI

Sun X., Guo L.-D. Endophytic fungal diversity: Review of traditional and molecular techniques. Mycology. 2012;3:65–76. doi: 10.1080/21501203.2012.656724. DOI

Hardoim Pablo R., van Overbeek Leonard S., Berg G., Pirttilä Anna M., Compant S., Campisano A., Döring M., Sessitsch A. The Hidden World within Plants: Ecological and Evolutionary Considerations for Defining Functioning of Microbial Endophytes. Microbiol. Mol. Biol. Rev. 2015;79:293–320. doi: 10.1128/MMBR.00050-14. PubMed DOI PMC

Liao C., Doilom M., Jeewon R., Hyde K.D., Manawasinghe I.S., Chethana K.W.T., Balasuriya A., Thakshila S.A.D., Luo M., Mapook A., et al. Challenges and update on fungal endophytes: Classification, definition, diversity, ecology, evolution and functions. Fungal Divers. 2025;131:301–367. doi: 10.1007/s13225-025-00550-5. DOI

Song Z., Kennedy P.G., Liew F.J., Schilling J.S. Fungal endophytes as priority colonizers initiating wood decomposition. Funct. Ecol. 2017;31:407–418. doi: 10.1111/1365-2435.12735. DOI

Baldrian P. Forest microbiome: Diversity, complexity and dynamics. FEMS Microbiol. Rev. 2017;41:109–130. doi: 10.1093/femsre/fuw040. PubMed DOI

Heilmann-Clausen J., Barron E.S., Boddy L., Dahlberg A., Griffith G.W., Nordén J., Ovaskainen O., Perini C., Senn-Irlet B., Halme P. A fungal perspective on conservation biology. Conserv. Biol. 2015;29:61–68. doi: 10.1111/cobi.12388. PubMed DOI

Hawksworth David L., Lücking R. Fungal Diversity Revisited: 2.2 to 3.8 Million Species. Microbiol. Spectr. 2017;5:10-1128. doi: 10.1128/microbiolspec.funk-0052-2016. PubMed DOI PMC

Lindahl B.D., Nilsson R.H., Tedersoo L., Abarenkov K., Carlsen T., Kjøller R., Kõljalg U., Pennanen T., Rosendahl S., Stenlid J., et al. Fungal community analysis by high-throughput sequencing of amplified markers—A user’s guide. New Phytol. 2013;199:288–299. doi: 10.1111/nph.12243. PubMed DOI PMC

Blackwell M. The Fungi: 1, 2, 3 … 5.1 million species? Am. J. Bot. 2011;98:426–438. doi: 10.3732/ajb.1000298. PubMed DOI

Toju H., Tanabe A.S., Yamamoto S., Sato H. High-Coverage ITS Primers for the DNA-Based Identification of Ascomycetes and Basidiomycetes in Environmental Samples. PLoS ONE. 2012;7:e40863. doi: 10.1371/journal.pone.0040863. PubMed DOI PMC

Nilsson R.H., Anslan S., Bahram M., Wurzbacher C., Baldrian P., Tedersoo L. Mycobiome diversity: High-throughput sequencing and identification of fungi. Nat. Rev. Microbiol. 2019;17:95–109. doi: 10.1038/s41579-018-0116-y. PubMed DOI

Tordoni E., Ametrano C.G., Banchi E., Ongaro S., Pallavicini A., Bacaro G., Muggia L. Integrated eDNA metabarcoding and morphological analyses assess spatio-temporal patterns of airborne fungal spores. Ecol. Indic. 2021;121:107032. doi: 10.1016/j.ecolind.2020.107032. DOI

Desprez-Loustau M.-L., Aguayo J., Dutech C., Hayden K.J., Husson C., Jakushkin B., Marçais B., Piou D., Robin C., Vacher C. An evolutionary ecology perspective to address forest pathology challenges of today and tomorrow. Ann. For. Sci. 2016;73:45–67. doi: 10.1007/s13595-015-0487-4. DOI

Eichmeier A., Spetik M., Frejlichova L., Pecenka J., Cechova J., Stefl L., Simek P. Survey of the Trunk Wood Mycobiome of an Ancient Tilia × europaea L. Appl. Microbiol. 2025;5:131. doi: 10.3390/applmicrobiol5040131. DOI

Sieber T.N. Endophytic fungi in forest trees: Are they mutualists? Fungal Biol. Rev. 2007;21:75–89. doi: 10.1016/j.fbr.2007.05.004. DOI

Terhonen E., Blumenstein K., Kovalchuk A., Asiegbu F.O. Forest Tree Microbiomes and Associated Fungal Endophytes: Functional Roles and Impact on Forest Health. Forests. 2019;10:42. doi: 10.3390/f10010042. DOI

Dawson S.K., Berglund H., Ovaskainen O., Jonsson B.G., Snäll T., Ottosson E., Jönsson M. Fungal trait-environment relationships in wood-inhabiting communities of boreal forest patches. Funct. Ecol. 2024;38:1944–1958. doi: 10.1111/1365-2435.14627. DOI

Jönsson M.T., Edman M., Jonsson B.G. Colonization and extinction patterns of wood-decaying fungi in a boreal old-growth Picea abies forest. J. Ecol. 2008;96:1065–1075. doi: 10.1111/j.1365-2745.2008.01411.x. DOI

Liao H.-L., Bonito G., Rojas J.A., Hameed K., Wu S., Schadt C.W., Labbé J., Tuskan G.A., Martin F., Grigoriev I.V., et al. Fungal Endophytes of Populus trichocarpa Alter Host Phenotype, Gene Expression, and Rhizobiome Composition. Mol. Plant-Microbe Interact.®. 2019;32:853–864. doi: 10.1094/MPMI-05-18-0133-R. PubMed DOI

Helander M., Ahlholm J., Sieber T.N., Hinneri S., Saikkonen K. Fragmented environment affects birch leaf endophytes. New Phytol. 2007;175:547–553. doi: 10.1111/j.1469-8137.2007.02110.x. PubMed DOI

Scholtysik A., Unterseher M., Otto P., Wirth C. Spatio-temporal dynamics of endophyte diversity in the canopy of European ash (Fraxinus excelsior) Mycol. Prog. 2013;12:291–304. doi: 10.1007/s11557-012-0835-9. DOI

Ihrmark K., Bödeker I.T.M., Cruz-Martinez K., Friberg H., Kubartova A., Schenck J., Strid Y., Stenlid J., Brandström-Durling M., Clemmensen K.E., et al. New primers to amplify the fungal ITS2 region–evaluation by 454-sequencing of artificial and natural communities. FEMS Microbiol. Ecol. 2012;82:666–677. doi: 10.1111/j.1574-6941.2012.01437.x. PubMed DOI

White T.J., Bruns T., Lee S., Taylor J. Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. In: Innis M.A., Gelfand D.H., Sninsky J.J., White T.J., editors. PCR Protocols. Academic Press; San Diego, CA, USA: 1990. pp. 315–322.

Stover N.A., Cavalcanti A.R.O. Using NCBI BLAST. Curr. Protoc. Essent. Lab. Tech. 2017;14:11.11.11–11.11.34. doi: 10.1002/cpet.8. DOI

Andrews S. FastQC: A Quality Control Tool for High Throughput Sequence Data. Babraham Bioinformatics, Babraham Institute; Cambridge, UK: 2010. [(accessed on 8 January 2026)]. Available online: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/

Větrovský T., Baldrian P., Morais D. SEED 2: A user-friendly platform for amplicon high-throughput sequencing data analyses. Bioinformatics. 2018;34:2292–2294. doi: 10.1093/bioinformatics/bty071. PubMed DOI PMC

Aronesty E. Comparison of Sequencing Utility Programs. Open Bioinform. J. 2013;7:1–8. doi: 10.2174/1875036201307010001. DOI

Bengtsson-Palme J., Ryberg M., Hartmann M., Branco S., Wang Z., Godhe A., De Wit P., Sánchez-García M., Ebersberger I., de Sousa F., et al. Improved software detection and extraction of ITS1 and ITS2 from ribosomal ITS sequences of fungi and other eukaryotes for analysis of environmental sequencing data. Methods Ecol. Evol. 2013;4:914–919. doi: 10.1111/2041-210X.12073. DOI

Blaxter M., Mann J., Chapman T., Thomas F., Whitton C., Floyd R., Abebe E. Defining operational taxonomic units using DNA barcode data. Philos. Trans. R. Soc. B Biol. Sci. 2005;360:1935–1943. doi: 10.1098/rstb.2005.1725. PubMed DOI PMC

Edgar R.C. UPARSE: Highly accurate OTU sequences from microbial amplicon reads. Nat. Methods. 2013;10:996–998. doi: 10.1038/nmeth.2604. PubMed DOI

Nilsson R.H., Larsson K.-H., Taylor A.F.S., Bengtsson-Palme J., Jeppesen T.S., Schigel D., Kennedy P., Picard K., Glöckner F.O., Tedersoo L., et al. The UNITE database for molecular identification of fungi: Handling dark taxa and parallel taxonomic classifications. Nucleic Acids Res. 2019;47:259–264. doi: 10.1093/nar/gky1022. PubMed DOI PMC

Baldrian P., Větrovský T., Lepinay C., Kohout P. High-throughput sequencing view on the magnitude of global fungal diversity. Fungal Divers. 2022;114:539–547. doi: 10.1007/s13225-021-00472-y. DOI

Tedersoo L., Bahram M., Põlme S., Kõljalg U., Yorou N.S., Wijesundera R., Ruiz L.V., Vasco-Palacios A.M., Thu P.Q., Suija A., et al. Global diversity and geography of soil fungi. Science. 2014;346:1256688. doi: 10.1126/science.1256688. PubMed DOI

Gloor G.B., Macklaim J.M., Pawlowsky-Glahn V., Egozcue J.J. Microbiome Datasets Are Compositional: And This Is Not Optional. Front. Microbiol. 2017;8:2224. doi: 10.3389/fmicb.2017.02224. PubMed DOI PMC

Greenacre M., Martínez-Álvaro M., Blasco A. Compositional Data Analysis of Microbiome and Any-Omics Datasets: A Validation of the Additive Logratio Transformation. Front. Microbiol. 2021;12:727398. doi: 10.3389/fmicb.2021.727398. PubMed DOI PMC

Hill M.O. Diversity and Evenness: A Unifying Notation and Its Consequences. Ecology. 1973;54:427–432. doi: 10.2307/1934352. DOI

Willis A.D. Rarefaction, Alpha Diversity, and Statistics. Front. Microbiol. 2019;10:2407. doi: 10.3389/fmicb.2019.02407. PubMed DOI PMC

Anderson M.J. Distance-Based Tests for Homogeneity of Multivariate Dispersions. Biometrics. 2006;62:245–253. doi: 10.1111/j.1541-0420.2005.00440.x. PubMed DOI

Nguyen N.H., Song Z., Bates S.T., Branco S., Tedersoo L., Menke J., Schilling J.S., Kennedy P.G. FUNGuild: An open annotation tool for parsing fungal community datasets by ecological guild. Fungal Ecol. 2016;20:241–248. doi: 10.1016/j.funeco.2015.06.006. DOI

Kohl M., Wiese S., Warscheid B. Cytoscape: Software for Visualization and Analysis of Biological Networks. In: Hamacher M., Eisenacher M., Stephan C., editors. Data Mining in Proteomics: From Standards to Applications. Humana Press; Totowa, NJ, USA: 2011. pp. 291–303. PubMed

Yang S., Poorter L., Kuramae E.E., Sass-Klaassen U., Leite M.F.A., Costa O.Y.A., Kowalchuk G.A., Cornelissen J.H.C., van Hal J., Goudzwaard L., et al. Stem traits, compartments and tree species affect fungal communities on decaying wood. Environ. Microbiol. 2022;24:3625–3639. doi: 10.1111/1462-2920.15953. PubMed DOI PMC

Krah F.-S., Bässler C., Heibl C., Soghigian J., Schaefer H., Hibbett D.S. Evolutionary dynamics of host specialization in wood-decay fungi. BMC Evol. Biol. 2018;18:119. doi: 10.1186/s12862-018-1229-7. PubMed DOI PMC

Menkis A., Redr D., Bengtsson V., Hedin J., Niklasson M., Nordén B., Dahlberg A. Endophytes dominate fungal communities in six-year-old veteranisation wounds in living oak trunks. Fungal Ecol. 2022;59:101020. doi: 10.1016/j.funeco.2020.101020. DOI

Cregger M.A., Veach A.M., Yang Z.K., Crouch M.J., Vilgalys R., Tuskan G.A., Schadt C.W. The Populus holobiont: Dissecting the effects of plant niches and genotype on the microbiome. Microbiome. 2018;6:31. doi: 10.1186/s40168-018-0413-8. PubMed DOI PMC

Wang Y., Zhang W., Ding C., Zhang B., Huang Q., Huang R., Su X. Endophytic Communities of Transgenic Poplar Were Determined by the Environment and Niche Rather Than by Transgenic Events. Front. Microbiol. 2019;10:588. doi: 10.3389/fmicb.2019.00588. PubMed DOI PMC

Spies C.F.J., Moyo P., Halleen F., Mostert L. Phaeoacremonium species diversity on woody hosts in the Western Cape Province of South Africa. Persoonia-Mol. Phylogeny Evol. Fungi. 2018;40:26–62. doi: 10.3767/persoonia.2018.40.02. PubMed DOI PMC

Travadon R., Lawrence D.P., Rooney-Latham S., Gubler W.D., Wilcox W.F., Rolshausen P.E., Baumgartner K. Cadophora species associated with wood-decay of grapevine in North America. Fungal Biol. 2015;119:53–66. doi: 10.1016/j.funbio.2014.11.002. PubMed DOI

Spetik M., Pecenka J., Stuskova K., Stepanova B., Eichmeier A., Kiss T. Fungal Trunk Diseases Causing Decline of Apricot and Plum Trees in the Czech Republic. Plant Dis. 2023;108:1425–1436. doi: 10.1094/PDIS-06-23-1080-SR. PubMed DOI

Damm U., Mostert L., Crous P.W., Fourie P.H. Novel Phaeoacremonium species associated with necrotic wood of Prunus trees. Persoonia-Mol. Phylogeny Evol. Fungi. 2008;20:87–102. doi: 10.3767/003158508X324227. PubMed DOI PMC

Argiroff William A., Carrell Alyssa A., Klingeman Dawn M., Dove Nicholas C., Muchero W., Veach Allison M., Wahl T., Lebreux Steven J., Webb Amber B., Peyton K., et al. Seasonality and longer-term development generate temporal dynamics in the Populus microbiome. mSystems. 2024;9:e00886-23. doi: 10.1128/msystems.00886-23. PubMed DOI PMC

Hiscox J., Savoury M., Müller C.T., Lindahl B.D., Rogers H.J., Boddy L. Priority effects during fungal community establishment in beech wood. ISME J. 2015;9:2246–2260. doi: 10.1038/ismej.2015.38. PubMed DOI PMC

Lin C.-P., Lin Y.-F., Liu Y.-C., Lu M.-Y.J., Ke H.-M., Tsai I.J. Spatiotemporal dynamics reveal high turnover and contrasting assembly processes in fungal communities across contiguous habitats of tropical forests. Environ. Microbiome. 2025;20:23. doi: 10.1186/s40793-025-00683-9. PubMed DOI PMC

Purahong W., Wubet T., Lentendu G., Hoppe B., Jariyavidyanont K., Arnstadt T., Baber K., Otto P., Kellner H., Hofrichter M., et al. Determinants of Deadwood-Inhabiting Fungal Communities in Temperate Forests: Molecular Evidence from a Large Scale Deadwood Decomposition Experiment. Front. Microbiol. 2018;9:2120. doi: 10.3389/fmicb.2018.02120. PubMed DOI PMC

Hoppe B., Purahong W., Wubet T., Kahl T., Bauhus J., Arnstadt T., Hofrichter M., Buscot F., Krüger D. Linking molecular deadwood-inhabiting fungal diversity and community dynamics to ecosystem functions and processes in Central European forests. Fungal Divers. 2016;77:367–379. doi: 10.1007/s13225-015-0341-x. DOI

Runnel K., Tedersoo L., Krah F.-S., Piepenbring M., Scheepens J.F., Hollert H., Johann S., Meyer N., Bässler C. Toward harnessing biodiversity–ecosystem function relationships in fungi. Trends Ecol. Evol. 2025;40:180–190. doi: 10.1016/j.tree.2024.10.004. PubMed DOI

Davis E.L., Weatherhead E., Koide R.T. The potential saprotrophic capacity of foliar endophytic fungi from Quercus gambelii. Fungal Ecol. 2023;62:101221. doi: 10.1016/j.funeco.2022.101221. DOI

Cline L.C., Schilling J.S., Menke J., Groenhof E., Kennedy P.G. Ecological and functional effects of fungal endophytes on wood decomposition. Funct. Ecol. 2018;32:181–191. doi: 10.1111/1365-2435.12949. DOI

Tanney J.B., Kemler M., Vivas M., Wingfield M.J., Slippers B. Silent invaders: The hidden threat of asymptomatic phytobiomes to forest biosecurity. New Phytol. 2025;247:533–545. doi: 10.1111/nph.70209. PubMed DOI PMC

Cosner J., Pandharikar G., Tremble K., Nash J., Rush T.A., Vilgalys R., Veneault-Fourrey C. Fungal endophytes. Curr. Biol. 2025;35:R904–R910. doi: 10.1016/j.cub.2025.08.058. PubMed DOI

Peay K.G., Kennedy P.G., Talbot J.M. Dimensions of biodiversity in the Earth mycobiome. Nat. Rev. Microbiol. 2016;14:434–447. doi: 10.1038/nrmicro.2016.59. PubMed DOI

Ma B., Wang H., Dsouza M., Lou J., He Y., Dai Z., Brookes P.C., Xu J., Gilbert J.A. Geographic patterns of co-occurrence network topological features for soil microbiota at continental scale in eastern China. ISME J. 2016;10:1891–1901. doi: 10.1038/ismej.2015.261. PubMed DOI PMC

Boddy L., Hiscox J. Fungal Ecology: Principles and Mechanisms of Colonization and Competition by Saprotrophic Fungi. Microbiol. Spectr. 2016;4:0019. doi: 10.1128/microbiolspec.FUNK-0019-2016. PubMed DOI

Toju H., Peay K.G., Yamamichi M., Narisawa K., Hiruma K., Naito K., Fukuda S., Ushio M., Nakaoka S., Onoda Y., et al. Core microbiomes for sustainable agroecosystems. Nat. Plants. 2018;4:247–257. doi: 10.1038/s41477-018-0139-4. PubMed DOI

Gao C., Xu L., Montoya L., Madera M., Hollingsworth J., Chen L., Purdom E., Singan V., Vogel J., Hutmacher R.B., et al. Co-occurrence networks reveal more complexity than community composition in resistance and resilience of microbial communities. Nat. Commun. 2022;13:3867. doi: 10.1038/s41467-022-31343-y. PubMed DOI PMC

Kitagami Y., Matsuda Y. Forest types matter for the community and co-occurrence network patterns of soil bacteria, fungi, and nematodes. Pedobiologia. 2024;107:151004. doi: 10.1016/j.pedobi.2024.151004. DOI

Nordén B., Andreasen M., Gran O., Menkis A. Fungal diversity in wood of living trees is higher in oak than in beech, maple or linden, and is affected by tree size and climate. Biodivers. Conserv. 2025;34:3609–3632. doi: 10.1007/s10531-025-03119-5. DOI