Symbioses with fungi are important and ubiquitous on dry land but underexplored in the sea. As yet only one seagrass has been shown to form a specific root-fungus symbiosis that resembles those occurring in terrestrial plants, namely the dominant long-lived Mediterranean species Posidonia oceanica (Alismatales: Posidoniaceae) forming a dark septate (DS) endophytic association with the ascomycete Posidoniomyces atricolor (Pleosporales: Aigialaceae). Using stereomicroscopy, light and scanning electron microscopy, and DNA cloning, here we describe a novel root-fungus symbiosis in the Indo-Pacific seagrass Thalassodendron ciliatum (Alismatales: Cymodoceaceae) from a site in the Gulf of Aqaba in the Red Sea. Similarly to P. oceanica, the mycobiont of T. ciliatum occurs more frequently in thinner roots that engage in nutrient uptake from the seabed and forms extensive hyphal mantles composed of DS hyphae on the root surface. Contrary to P. oceanica, the mycobiont occurs on the roots with root hairs and does not colonize its host intraradically. While the cloning revealed a relatively rich spectrum of fungi, they were mostly parasites or saprobes of uncertain origin and the identity of the mycobiont thus remains unknown. Symbioses of seagrasses with fungi are probably more frequent than previously thought, but their functioning and significance are unknown. Melanin present in DS hyphae slows down their decomposition and so is true for the colonized roots. DS fungi may in this way conserve organic detritus in the seagrasses' rhizosphere, thus contributing to blue carbon sequestration in seagrass meadows.

Department of Mycorrhizal Symbioses Institute of Botany Czech Academy of Sciences Průhonice Czechia

KROKODIVE CZ Údolní 219 47 Prague 14700 Czechia

Laboratory of Molecular Biology and Bioinformatics Institute of Botany Czech Academy of Sciences Lesní 322 Průhonice 25243 Czechia

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Agerer R (1987) Colour atlas of ectomycorrhizae. Einhorn, Schwäbisch Gmünd

Amend A (2014) From dandruff to deep-sea vents: Malassezia-like fungi are ecologically hyper-diverse. PLoS Pathog 10:e1004277. 10.1371/journal.ppat.1004277 PubMed PMC

Angiosperm Phylogeny Website (2023) http://www.mobot.org/MOBOT/research/APweb/welcome.html, accessed 30/8/2023

Arnold AE (2007) Understanding the diversity of foliar endophytic fungi: progress, challenges, and frontiers. Fungal Biol Rev 21:51–66. 10.1016/j.fbr.2007.05.003

Badalamenti F, Alagna A, Fici S (2015) Evidences of adaptive traits to rocky substrates undermine paradigm of habitat preference of the Mediterranean seagrass Posidonia oceanica. Sci Rep 5:8804. 10.1038/srep08804 PubMed PMC

Boddy L, Watkinson SC (1995) Wood decomposition, higher fungi, and their role in nutrient redistribution. Can J Bot 73:1377–1383. 10.1139/b95-400

Borovec O, Vohník M (2018) Ontogenetic transition from specialized root hairs to specific root-fungus symbiosis in the dominant Mediterranean seagrass Posidonia oceanica. Sci Rep 8:1–11. 10.1038/s41598-018-28989-4 PubMed PMC

Brundrett MC (2002) Coevolution of roots and mycorrhizas of land plants. New Phytol 154:275–304. 10.1046/j.1469-8137.2002.00397.x PubMed

Brundrett MC (2017) Global diversity and importance of mycorrhizal and nonmycorrhizal plants, in: Tedersoo L (ed) Biogeography of mycorrhizal symbiosis. Springer International Publishing. 10.1007/978-3-319-56363-3_21

Cariello L, Zanetti L, De Stefano S (1979) Posidonia ecosystem – V. Phenolic compounds from marine phanerogames, Cymodocea nodosa and Posidonia oceanica. Comp Biochem Physiol B Biochem Mol Biol 62B:159–161. 10.1016/0305-0491(79)90304-3

Domínguez ER (2017) Crocicreas gramineum var. gramineum. https://www.centrodeestudiosmicologicosasturianos.org/?p=725, accessed 2/9/2023

Ducomet V (1907) Recherches sur le développement de quelques champignons parasites à Thalle subcuticulaire. Thèse Doctorale. Faculté des sciences de Paris, Rennes

Ekanayaka A, Hyde K, Gentekaki E, McKenzie E, Zhao Q et al (2019) Preliminary classification of Leotiomycetes. Mycosphere 10:310–489. 10.5943/mycosphere/10/1/7

Elliott JK, Simpson H, Teesdale A, Replogle A, Elliott MG et al (2019) A novel phagomyxid parasite produces sporangia in root hair galls of eelgrass (Zostera marina). Protist 170:64–81. 10.1016/j.protis.2018.12.001 PubMed

Ettinger CL, Eisen JA (2019) Characterization of the mycobiome of the seagrass, Zostera marina, reveals putative associations with marine chytrids. Front Microbiol 10:2476. 10.3389/fmicb.2019.02476 PubMed PMC

Fehrer J, Réblová M, Bambasová V, Vohník M (2019) The root-symbiotic Rhizoscyphus ericae aggregate and Hyaloscypha (Leotiomycetes) are congeneric: phylogenetic and experimental evidence. Stud Mycol 92:195–225. 10.1016/j.simyco.2018.10.004 PubMed PMC

Fourqurean JW, Duarte CM, Kennedy H, Marbà N, Holmer M et al (2012) Seagrass ecosystems as a globally significant carbon stock. Nat Geosci 5:505–509. 10.1038/ngeo1477

Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118. 10.1111/j.1365-294X.1993.tb00005.x PubMed

Green EP, Short FT (2003) World atlas of seagrasses. University of California Press, USA

Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98

Hawkins H-J, Cargill RIM, van Nuland ME, Hagen SC, Field KJ et al (2023) Mycorrhizal mycelium as a global carbon pool. Curr Biol 33:R560–R573. 10.1016/j.cub.2023.02.027 PubMed

Hongsanan S, Sánchez-Ramírez S, Crous PW, Ariyawansa HA et al (2016) The evolution of fungal epiphytes. Mycosphere 7:1690–1712. 10.5943/mycosphere/7/11/6

Hyde KD, Jones EBG, Liu J-K, Ariyawansa H, Boehm E et al (2013) Families of Dothideomycetes. Fungal Divers 63:1–313. 10.1007/s13225-013-0263-4

Jayasiri SC, Hyde KD, Ariyawansa HA, Bhat J, Buyck B et al (2015) The Faces of Fungi database: fungal names linked with morphology, phylogeny and human impacts. Fungal Divers 74:3–18. 10.1007/s13225-015-0351-8

Jumpponen A (2001) Dark septate endophytes – are they mycorrhizal? Mycorrhiza 11:207–211. 10.1007/s005720100112

Jumpponen A, Trappe JM (1998) Dark septate endophytes: a review of facultative biotrophic root-colonizing fungi. New Phytol 140:295–310 PubMed

Kaldorf M, Renker C, Fladung M, Buscot F (2004) Characterization and spatial distribution of ectomycorrhizas colonizing aspen clones released in an experimental field. Mycorrhiza 14:295–306. 10.1007/s00572-003-0266-1 PubMed

Kariman K, Barker SJ, Jost R, Finnegan PM, Tibbett M (2014) A novel plant–fungus symbiosis benefits the host without forming mycorrhizal structures. New Phytol 201:1413–1422. 10.1111/nph.12600 PubMed

Kohout P, Sýkorová Z, Čtvrtlíková M, Rydlová J, Suda J et al (2012) Surprising spectra of root-associated fungi in submerged aquatic plants. FEMS Microbiol Ecol 80:216–235. 10.1111/j.1574-6941.2011.01291.x PubMed

Kolařík M, Vohník M (2018) When the ribosomal DNA does not tell the truth: the case of the taxonomic position of Kurtia Argillacea, an ericoid mycorrhizal fungus residing among Hymenochaetales. Fungal Biol 122:1–18. 10.1016/j.funbio.2017.09.006 PubMed

Kolátková V, Vohník M (2019) Adaptive traits in the seagrass Posidonia oceanica: Root hairs with spiral cell walls, not spiral root hairs. Aquat Bot 155:52–53. 10.1016/j.aquabot.2018.11.013

Kolátková V, Mooney M, Kelly K, Hineva E, Gawryluk RMR et al. (2023) Eelgrass (Zostera spp.) associated phytomyxids are host-specific congeneric parasites and predominant eukaryotes in the eelgrass rhizosphere on a global scale. Environ Microbiol 25:1522–1537. 10.1111/1462-2920.16376 PubMed

Kumar S, Abedin MM, Singh AK, Das S (2020) Role of phenolic compounds in plant-defensive mechanisms. In: Lone R, Shuab R, Kamili A (eds) Plant phenolics in sustainable agriculture. Springer, Singapore. 10.1007/978-981-15-4890-1_22

Langley JA, Chapman SK, Hungate BA (2006) Ectomycorrhizal colonization slows root decomposition: the post-mortem fungal legacy. Ecol Lett 9:955–959. 10.1111/j.1461-0248.2006.00948.x PubMed

Larkum AWD, Waycott M, Conran JG (2018) Evolution and biogeography of seagrasses. in: Larkum AWD, Kendrick GA, Ralph PJ (2018) Seagrasses of Australia. Springer, Cham. 10.1007/978-3-319-71354-0_1

Lawrey JD, Diederich P (2018) Lichenicolous fungi: worldwide checklist, including isolated cultures and sequences available. http://www.lichenicolous.net, accessed 1/9/2023

Le Renard L, Stockey RA, Upchurch GR, Berbee ML (2021) Extending the fossil record for foliicolous Dothideomycetes: Bleximothyrium ostiolatum gen. et sp. nov., a unique fly-speck fungus from the Lower Cretaceous of Virginia, USA. Am J Bot 108:129–144. 10.1002/ajb2.1602 PubMed

Lefebvre L, Compère P, Gobert S (2023) The formation of aegagropiles from the Mediterranean seagrass Posidonia oceanica (L.) Delile (1813): plant tissue sources and colonisation by melanised fungal mycelium. Mar Biol 170:19. 10.1007/s00227-022-04166-0

Lipkin Y (1979) Quantitative aspects of seagrass communities, particularly of those dominated by Halophila stipulacea, in Sinai (Northern Red Sea). Aquat Bot 7:119–128. 10.1016/0304-3770(79)90016-0

Lukešová T, Kohout P, Větrovský T, Vohník M (2015) The potential of dark septate endophytes to form root symbioses with ectomycorrhizal and ericoid mycorrhizal middle European forest plants. PLoS ONE 10:e0124752. 10.1371/journal.pone.0124752 PubMed PMC

Mata JL, Cebrián J (2013) Fungal endophytes of the seagrasses Halodule wrightii and Thalassia testudinum in the northcentral Gulf of Mexico. Bot Mar 56:541–545. 10.1515/bot-2013-0047

Mayerhofer MS, Kernaghan G, Harper KA (2013) The effects of fungal root endophytes on plant growth: a meta-analysis. Mycorrhiza 23:119–128. 10.1007/s00572-012-0456-9 PubMed

McMillan C (1984) The condensed tannins (proanthocyanidins) in seagrasses. Aquat Bot 20:351–357. 10.1016/0304-3770(84)90099-8

Naranjo-Ortiz MA, Gabaldón T (2019) Fungal evolution: major ecological adaptations and evolutionary transitions. Biol Rev 94:1443–1476. 10.1111/brv.12510 PubMed PMC

Newsham KK (1999) Phialophora graminicola, a dark septate fungus, is a beneficial associate of the grass Vulpia ciliata ssp. ambigua. New Phytol 144:517–524. 10.1046/j.1469-8137.1999.00537.x PubMed

Newsham KK (2011) A meta-analysis of plant responses to dark septate root endophytes. New Phytol 190:783–793. 10.1111/j.1469-8137.2010.03611.x PubMed

Nguyen NH, Song Z, Bates ST, Branco S, Tedersoo L et al (2016) FUNGuild: an open annotation tool for parsing fungal community datasets by ecological guild. Fungal Ecol 20:241–248. 10.1016/j.funeco.2015.06.006

Nilsson RH, Larsson K-H, Taylor AFS, Bengtsson-Palme J, Jeppesen TS et al (2019) The UNITE database for molecular identification of fungi: handling dark taxa and parallel taxonomic classifications. Nucleic Acids Res 47:D259–D264. 10.1093/nar/gky1022 PubMed PMC

Osono T (2007) Ecology of ligninolytic fungi associated with leaf litter decomposition. Ecol Res 22:955–974. 10.1007/s11284-007-0390-z

Parniske M (2008) Arbuscular mycorrhiza: the mother of plant root endosymbioses. Nat Rev Microbiol 6:763–775. 10.1038/nrmicro1987 PubMed

Piñeiro-Juncal N, Leiva-Dueñas C, Serrano O, Mateo MÁ, Martínez-Cortízas A (2020) Pedogenic processes in a Posidonia oceanica mat. Soil Syst 4:18. 10.3390/soilsystems4020018

Podgórska-Kryszczuk I, Solarska E, Kordowska-Wiater M (2022) Biological control of Fusarium culmorum, Fusarium graminearum and Fusarium poae by antagonistic yeasts. Pathogens 11:86. 10.3390/pathogens11010086 PubMed PMC

Poli A, Prigione V, Bovio E, Perugini I, Varese GC (2021) Insights on Lulworthiales inhabiting the Mediterranean Sea and description of three novel species of the genus Paralulworthia. J Fungi 7:940. 10.3390/jof7110940 PubMed PMC

Powley HR, Cappellen PV, Krom MD (2017) Nutrient cycling in the Mediterranean Sea: The key to understanding how the unique marine ecosystem functions and responds to anthropogenic pressures. in: Mediterranean Identities - Environment, Society, Culture. InTech. 10.5772/intechopen.70878

Read DJ, Leake JR, Perez-Moreno J (2004) Mycorrhizal fungi as drivers of ecosystem processes in heathland and boreal forest biomes. Can J Bot 82:1243–1263. 10.1139/B04-123

Reininger V, Sieber TN Mycorrhiza reduces adverse effects of dark septate endophytes (DSE) on growth of conifers. PLoS ONE 7:e0042865. 10.1371/journal.pone.0042865 PubMed PMC

Rice AV, Currah RS (2006) Oidiodendron maius: Saprobe in Sphagnum peat, mutualist in ericaceous roots? In: Schulz BJE, Boyle CJC, Sieber TN (eds) Microbial Root endophytes, vol 9. Springer, Berlin. 10.1007/3-540-33526-9_13

Saadatzadeh MR, Ashbee HR, Holland KT, Ingham E (2001) Production of the mycelial phase of Malassezia in vitro. Med Mycol 39:487–493. 10.1080/714031063 PubMed

Saikkonen K, Faeth SH, Helander M, Sullivan TJ (1998) Fungal endophytes: a continuum of interactions with host plants. Annu Rev Ecol Syst 29:319–343. 10.1146/annurev.ecolsys.29.1.319

Sakayaroj J, Preedanon S, Supaphon Jones EBG, Phongpaichit S (2010) Phylogenetic diversity of endophyte assemblages associated with the tropical seagrass Enhalus acoroides in Thailand. Fungal Divers 42:27–45. 10.1007/s13225-009-0013-9

Sayers EW, Cavanaugh M, Clark K, Ostell J, Pruitt KD, Karsch-Mizrachi I (2019) GenBank. Nucleic Acids Res 47:D94–D99. 10.1093/nar/gky989 PubMed PMC

Selosse M-A, Le Tacon F (1998) The land flora: a phototroph–fungus partnership? Trends Ecol Evol 13:15–20. 10.1016/S0169-5347(97)01230-5 PubMed

Selosse M, Vohník M, Chauvet E (2008) Out of the rivers: are some aquatic hyphomycetes plant endophytes? New Phytol 178:3–7. 10.1111/j.1469-8137.2008.02390.x PubMed

Serrano O, Mateo MA, Renom P, Julià R (2012) Characterization of soils beneath a Posidonia oceanica meadow. Geoderma 185–186:26–36. 10.1016/j.geoderma.2012.03.020

Short FT, McRoy CP (1984) Nitrogen uptake by leaves and roots of the seagrass Zostera marina L. Bot Mar 27:547–555. 10.1515/botm.1984.27.12.547

Shrestha P, Szaro TM, Bruns TD, Taylor JW (2011) Systematic search for cultivatable fungi that best deconstruct cell walls of Miscanthus and sugarcane in the field. Appl Environ Microbiol 77:5490–5504. 10.1128/AEM.02996-10 PubMed PMC

Singh SM, Tsuji M, Gawas-Sakhalker P, Loonen MJJE, Hoshino T (2016) Bird feather fungi from Svalbard Arctic. Polar Biol 39:523–532. 10.1007/s00300-015-1804-y

Solomon PS, Lowe RGT, Tan K-C, Waters ODC, Oliver RP (2006) Stagonospora nodorum: cause of Stagonospora nodorum blotch of wheat. Mol Plant Pathol 7:147–156. 10.1111/j.1364-3703.2006.00326.x PubMed

Špetík M, Berraf-Tebbal A, Pokluda R, Eichmeier A (2021) Pyrenochaetopsis kuksensis (Pyrenochaetopsidaceae), a new species associated with an ornamental boxwood in the Czech Republic. Phytotaxa 498:177–185. 10.11646/phytotaxa.498.3.3

Stenglein SA (2009) Fusarium poae: a pathogen that needs more attention. J Plant Pathol 91:25–36. 10.4454/jpp.v91i1.621

Suetrong S, Schoch CL, Spatafora JW, Kohlmeyer J, Volkmann-Kohlmeyer B et al (2009) Molecular systematics of the marine Dothideomycetes. Stud Mycol 64:155–173. 10.3114/sim.2009.64.09 PubMed PMC

Terrados J, Williams SL (1997) Leaf versus root nitrogen uptake by the surfgrass Phyllospadix torreyi. Mar Ecol Prog Ser 149:267–277. https://www.int-res.com/abstracts/meps/v149/p267-277

Torta L, Burruano S, Giambra S, Conigliaro G, Piazza G et al. (2022) Cultivable fungal endophytes in roots, rhizomes and leaves of Posidonia oceanica (L.) Delile along the coast of Sicily, Italy. Plants 11:1139. 10.3390/plants11091139 PubMed PMC

Usuki F, Narisawa K (2007) A mutualistic symbiosis between a dark septate endophytic fungus, Heteroconium chaetospira, and a nonmycorrhizal plant, Chinese cabbage. Mycologia 99:175–184. 10.3852/mycologia.99.2.175 PubMed

van der Heijden MGA, Martin FM, Selosse MA, Sanders IR (2015) Mycorrhizal ecology and evolution: the past, the present, and the future. New Phytol 205:1406–1423. 10.1111/nph.13288 PubMed

Vohník M (2020) Ericoid mycorrhizal symbiosis: theoretical background and methods for its comprehensive investigation. Mycorrhiza 30:671–695. 10.1007/s00572-020-00989-1 PubMed PMC

Vohník M (2021) Bioerosion and fungal colonization of the invasive foraminiferan Amphistegina lobifera in a Mediterranean seagrass meadow. Biogeosciences 18:2777–2790. 10.5194/bg-18-2777-2021

Vohník M (2022) Are lulworthioid fungi dark septate endophytes of the dominant Mediterranean seagrass Posidonia oceanica? Plant Biol 24:127–133. 10.1111/plb.13353 PubMed

Vohník M, Réblová M (2023) Fungi in hair roots of Vaccinium spp. (Ericaceae) growing on decomposing wood: colonization patterns, identity, and in vitro symbiotic potential. Mycorrhiza 33:69–86. 10.1007/s00572-023-01101-z PubMed PMC

Vohník M, Lukančič S, Bahor E, Regvar M, Vosátka M et al (2003) Inoculation of Rhododendron cv. Belle-Heller with two strains of Phialocephala fortinii in two different substrates. Folia Geobot 38:191–200. 10.1007/BF02803151

Vohník M, Sadowsky JJ, Kohout P, Lhotáková Z, Nestby R, Kolařík M (2012) Novel root-fungus symbiosis in Ericaceae: sheathed ericoid mycorrhiza formed by a hitherto undescribed basidiomycete with affinities to Trechisporales. PLoS ONE 7:e39524. 10.1371/journal.pone.0039524 PubMed PMC

Vohník M, Borovec O, Župan I, Vondrášek D, Petrtýl M et al (2015) Anatomically and morphologically unique dark septate endophytic association in the roots of the Mediterranean endemic seagrass Posidonia oceanica. Mycorrhiza 25:663–672. 10.1007/s00572-015-0642-7 PubMed

Vohník M, Borovec O, Kolařík M (2016) Communities of cultivable root mycobionts of the seagrass Posidonia oceanica in the northwest Mediterranean Sea are dominated by a hitherto undescribed pleosporalean dark septate endophyte. Microb Ecol 71:442–451. 10.1007/s00248-015-0640-5 PubMed

Vohník M, Borovec O, Župan I, Kolařík M, Sudová R (2017) Fungal root symbionts of the seagrass Posidonia oceanica in the central Adriatic Sea revealed by microscopy, culturing and 454-pyrosequencing. Mar Ecol Prog Ser 583:107–120. 10.3354/meps12337

Vohník M, Borovec O, Kolaříková Z, Sudová R, Réblová M (2019) Extensive sampling and high-throughput sequencing reveal Posidoniomyces atricolor gen. et sp. nov. (Aigialaceae, Pleosporales) as the dominant root mycobiont of the dominant Mediterranean seagrass Posidonia oceanica. MycoKeys 55:59–86. 10.3897/mycokeys.55.35682 PubMed PMC

Wang G, Cao X, Ma X, Guo M, Liu C et al (2016) Diversity and effect of Trichoderma spp. associated with green mold disease on Lentinula edodes in China. Microbiologyopen 5:709–718. 10.1002/mbo3.364 PubMed PMC

White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. in: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR Protocols. Elsevier. 10.1016/B978-0-12-372180-8.50042-1

Williams J, Clarkson JM, Mills PR, Cooper RM (2003) Saprotrophic and mycoparasitic components of aggressiveness of Trichoderma harzianum groups toward the commercial mushroom Agaricus bisporus. Appl Environ Microbiol 69:4192–4199. 10.1128/AEM.69.7.4192-4199.2003 PubMed PMC

Yu T, Nassuth A, Peterson RL (2001) Characterization of the interaction between the dark septate fungus Phialocephala fortinii and Asparagus officinalis roots. Can J Microbiol 47:741–753. 10.1139/cjm-47-8-741 PubMed

Zhang Z, Schwartz S, Wagner L, Miller W (2000) A greedy algorithm for aligning DNA sequences. J Comput Biol 7:203–214. 10.1089/10665270050081478 PubMed

Zijlstra JD, Van’t Hof P, Baar J, Verkley GJM, Summerbell RC et al (2005) Diversity of symbiotic root endophytes of the Helotiales in ericaceous plants and the grass, Deschampsia flexuosa. Stud Mycol 53:147–162. 10.3114/sim.53.1.147