Prior to this study, only one seagrass has been reported to form a specific root-fungus symbiosis resembling those commonly occurring on dry land, and our observations thus extend the distribution and host taxonomic range of these associations for the NE Red Sea and another species in another seagrass family, respectively. However, unlike P. oceanica that is endemic to the Mediterranean, T. ciliatum is distributed across the Indo-Pacific (Green and Short, 2003), making its symbiosis a potentially widespread phenomenon. The same is true for the more speciose Cymodoceaceae vs. Posidoniaceae that occur in the Caribbean, NW Africa, the Mediterranean, and most of the Indo-Pacific vs. being limited to the Mediterranean and SW to SE Australia (Angiosperm Phylogeny Website 2023). On the other hand, it is not known whether other members of Cymodoceaceae and Posidoniaceae form similar root-fungus symbioses. For example, despite that Cymodocea nodosa often co-occurs with P. oceanica and belongs to the same family as T. ciliatum, it does not seem to form any specific root-fungus symbiosis (Vohník et al., 2015).
Root-fungus symbioses in T. ciliatum and P. oceanica
In addition to the differences in their distribution and taxonomy as well as anatomy and morphology of their roots, T. ciliatum and P. oceanica to some extent differ in anatomy and morphology of their root-fungus symbioses (Table 2). The most surprising difference is the absence of any visible intraradical hyphae in T. ciliatum, because in P. oceanica fungal hyphae often vigorously develop within the hypodermis (Vohník et al., 2015, 2019), forming the intracellular microsclerotia characteristic of DSE (e.g., Lukešová et al., 2015; Yu et al., 2001). In addition, while in P. oceanica fungal hyphae infrequently colonize the rhizodermal cells, these are fungus-free and filled with what appears as polyphenolic substances in T. ciliatum (cf. Cariello et al., 1979; McMillan, 1984). Lastly, in T. ciliatum the DS fungal mantles cover the basal parts of the root hairs, a trait to our knowledge unknown in terrestrial roots, while these are typically absent in P. oceanica roots colonized by Pos. atricolor (Borovec and Vohník, 2018). On the other hand, the mycobionts of both seagrasses form extensive hyphal mantles on the root surface (Vohník, 2022; Vohník et al., 2017), this study) that are morphologically identical to those formed by DSE and certain ectomycorrhizal (EcM) fungi on the roots of compatible terrestrial plants (e.g., Kaldorf et al., 2004).
Table 2
Comparison of the seagrasses Thalassodendron ciliatum and Posidonia oceanica with focus on their interactions with fungi
Seagrass species (family in Alismatales) | Distribution | Hypodermis | Root hairs | Tannin cells in the roots | Main fungal partner | Other fungal partners | Surface hyphal mantles | Intraradical colonization | Fungal interaction with root hairs |
Thalassodendron ciliatum (Cymodoceaceae) | Indo-Pacific | no | yes, often abundant in adults | yes (in rhizodermis) | unknown | see Table 1 | yes | no | dense hyphal mantles covering the root hairs’ bases |
Posidonia oceanica (Posidoniaceae) | Mediterranean Sea (endemic, remaining Posidonia species in southern Australia) | yes | abundant in seedlings, mostly absent in adults | no | Posidoniomyces atricolor (Aigialaceae, Pleosporales) | lulworthioid fungi (Lulworthiales), other marine fungi (see Introduction for references) | yes | yes (intracellular microsclerotia in hypodermis, intracellular hyphae in rhizodermis, intercellular hyphae in rhizodermis and hypodermis) | negative correlation with the root hairs’ presence |
Fungal partners in T. ciliatum and P. oceanica
It has been repeatedly shown that Pos. atricolor mycelium develops from the intracellular microsclerotia occurring in the hypodermis of P. oceanica (Vohník et al., 2016, 2019; Vohník, 2021, 2022) and Pos. atricolor has been detected in the terminal roots of P. oceanica adults at every sampled locality in the whole N Mediterranean (M. Vohník, unpublished data). At the same time, Pos. atricolor has not been detected in any other host or substrate nor by any other research team. In addition, the mycobiota of P. oceanica roots typically comprises lulworthioid fungi (Lulworthiales) (Torta et al., 2022; Poli et al., 2021; Vohník et al., 2016, 2017) but their functioning is unclear (Vohník, 2022). To our surprise, none of these fungi nor their relatives were detected in the investigated T. ciliatum roots. This might be due to their genuine absence, the different detection methods used in this (cloning) and the previous (culturing and high-throughput sequencing) studies, or incompatibility with the primers used in this study (cf. (Vohník et al., 2012).
To our knowledge, this is the first report on the mycobiota associated with the roots of T. ciliatum. In general, the most surprising results were the relatively high incidence of basidiomycetes and the dominance of saprotrophs and pathotrophs, both to a large extent due to the high incidence of Malassezia spp. (Table 1). Malassezia are ecologically versatile yeasts known from both terrestrial and marine environments and they occur on such diverse substrates as corals, deep-sea vents, and mammal skin (e.g., Amend, 2014). They are commensals, pathogens, and saprobes and only rarely form hyphae (e.g., Saadatzadeh et al., 2001). It is thus not probable that they form the DS hyphal mantles characteristic of the novel root-fungus symbiosis reported here. Similarly, none of the six non-Malassezia OTU with ≥ 3 sequences seem like probable candidates for the observed colonization pattern. For example, Fusarium poae is a known plant pathogen (e.g., Stenglein, 2009), Trichoderma are mycoparasites, saprobes, and pathogens (e.g., Williams et al., 2003) and none of them typically produce melanized hyphae (Podgórska-Kryszczuk et al., 2022; Wang et al., 2016).
Four OTU belonged to Pleosporales but none to Aigialaceae, i.e., the same family as Pos. atricolor. OTU-6/Pleosporales sp. grouped with Stagonospora sp. (GenBank OM337558, Massarinaceae), Phaeosphaeriopsis sp. (HQ630983, Phaeosphaeriaceae) obtained from Miscanthus giganteus (Poales: Poaceae) from Illinois, USA (Shrestha et al., 2011), and Didymocyrtis cladoniicola (LT796877, Phaeosphaeriaceae) from USA, all with > 99% sequence similarity. Stagonospora are probable plant pathogens (e.g., Solomon et al., 2006), Phaeosphaeriaceae are pathogenic, saprobic, or hyperparasitic mostly on monocotyledons and especially Poaceae (Hyde et al., 2013)d cladoniicola is a probable lichen parasite (Lawrey and Diederich, 2018). While no GenBank entry displayed > 90% sequence similarity with OTU-7/Pleosporales sp., OTU 25 belongs to Stagonospora sp. and OTU 26 to Pyrenochaetopsis sp. (Pyrenochaetopsidaceae), displaying 99.4% sequence similarity with Pyrenochaetopsis sp. PG293 (AB916515) from a bird feather from Svalbard (Singh et al., 2016). Pyrenochaetopsis comprises commensals, plant endophytes and pathogens, and saprobes occurring in animals, humans, plants, soil, and water (e.g., Špetík et al., 2021).
When searching for the mycobiont forming the novel symbiosis one should not discriminate fungi related to known saprobes and/or pathogens. For example, Pos. atricolor represents the only biotrophic lineage within the otherwise saprobic Aigialaceae (Vohník et al., 2019; Suetrong et al., 2009), certain mycorrhizal fungi also inhabit the soil and wood as saprobes (Rice and Currah, 2006; Fehrer et al., 2019; Kolařík and Vohník, 2018; Vohník and Réblová, 2023), etc. Likewise, not all fungi belonging to genera, families, and orders comprising widespread plant endophytes necessarily share this trait, an excellent example being Helotiales (e.g., Zijlstra et al., 2005). In our study, five OTU belonged to Helotiales: OTU 5 and 18 displayed affinities to Crocicreas gramineum (Helotiaceae) which is a saprobe on plant debris and leaves, especially on Poaceae (Domínguez 2017). OTU 20 clustered with several Lemonniera sp. (Discinellaceae) that are saprotrophs on dead plant material (Ekanayaka et al., 2019). Finally, OTU 21 and 22 belonged to Tetracladium (Helotiales inc. sed.) which comprises aquatic hyphomycetes sometimes colonizing plant roots as endophytes (Selosse et al., 2008). Under these circumstances, we cannot be sure if we detected the mycobiont forming the novel symbiosis nor what is its taxonomy. Nevertheless, despite the limited sampling our study reveals a relatively high fungal diversity associated with the roots of a common Indo-Pacific seagrass that begs further investigation, a situation similar to many freshwater plants (e.g., Kohout et al., 2012).
Functioning of DS fungal associations in seagrasses
There is an ongoing debate about the role of DSE in plant ecology and physiology and it seems that they can be beneficial, neutral, or detrimental associates of terrestrial plants, depending on the phytobiont and mycobiont taxonomy and ontogeny as well as a wide array of environmental conditions (Newsham, 2011; Reininger and Sieber, 2012; Usuki and Narisawa, 2007; Vohník et al., 2003; Mayerhofer et al., 2013). On the other hand, virtually nothing is known about the functioning of DSE/DS mycobionts in seagrasses and changing this will require manipulative monoxenic inoculation experiments, isotopic studies, and genome analyses. In P. oceanica, there is an ontogenetic shift from seedlings whose roots possess dense root hairs but lack the DSE symbiosis to adults mostly without root hairs but regularly forming the DSE symbiosis, which is similar to non-mycorrhizal vs. EcM roots (i.e., those colonized by EcM fungi) of EcM plants (Borovec and Vohník, 2018). However, it is unknown whether this shift is directly related to Pos. atricolor and in T. ciliatum, the mycobiont’s presence does not seem to be in any relationship with the presence of the root hairs.
Although indirect, this study provides two important hints on the functioning of the novel symbiosis in T. ciliatum. First, the observation that the hyphal mantles stay on the root surface without visible intraradical colonization suggests that the mycobiont lives as a fungal epiphyte. Epiphytism in fungi is an ancient widespread trait that has evolved independently in several ascomycetous lineages (Hongsanan et al., 2016) but typically concerns plant aboveground organs, especially the leaves, and to our knowledge has never been reported from the roots. While it is unclear whether any parallels can be drawn between terrestrial leaf and marine root fungal epiphytes, they might protect the roots from bacterial, fungal, and viral pathogens, damage caused by herbivores, osmotic stress, etc. In this context, it is interesting to note that older roots typically without fungal colonization had their rhizodermal cells filled with light- to dark brown structures of varied shapes, possibly formed by polyphenolic substances that protect the roots from the stresses listed above (Kumar et al., 2020). Since these were less intense in the colonized roots, one might hypothesize that the hyphal mantles take over their protection role, eventually saving the seagrass the energy and metabolites necessary to produce these substances. Second, the DS fungal colonization was more frequent in thinner terminal roots that are typically the sites of nutrient uptake, indicating a possible role of the mycobiont in the seagrass nutrition, as already hypothesized for Pos. atricolor in the dominant Mediterranean seagrass P. oceanica (Vohník et al., 2015). However, further research is needed to test these hypotheses.