# Root-associated Helotiales fungi: Overlooked players in plant nutrition

Pauline Bruyant, Yvan Moënne-Loccoz, Juliana Almario

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Pauline Bruyant, Yvan Moënne-Loccoz, Juliana Almario. Root-associated Helotiales fungi: Overlooked players in plant nutrition. Soil Biology and Biochemistry, 2024, 191, pp.109363. 10.1016/j.soilbio.2024.109363. hal-04662162

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Submitted on 25 Jul 2024

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Root-associated Helotiales fungi: Overlooked players in plant nutrition

Pauline Bruyant, Yvan Moënne-Loccoz, Juliana Almario*

Université Claude Bernard Lyon 1, Laboratoire d'Ecologie Microbienne, UMR CNRS 5557, UMR INRAE 1418, VetAgro Sup, 69622, Villeurbanne, France

A B S T R A C T

Fungi contribute to plant mineral nutrition through diverse symbiotic mycorrhizal associations. Although historically most attention has been put on Glomer-
omycotina fungi establishing the ancestral arbuscular mycorrhizal symbiosis, other fungal lineages have developed similar but evolutionarily younger associations
with plants. The Helotiales, a largely understudied fungal order, is emerging as a key lineage in fungus-mediated nutrient acquisition by plants. Indeed, these fungi
can form ericoid mycorrhizae or ectomycorrhizae while some isolates behave as so-called "mycorrhizal-like" fungi capable of transferring nutrients to their hosts in a
non-symbiotic way, i.e. without forming differentiated cellular structures inside plant roots, and without proven nutritional reciprocity from the plant. Here, we
explore current knowledge about the phylogeny of these plant-beneficial Helotiales and cover recent advances on the roles they play in plant nutrition, highlighting
the potential of these associations as alternatives to the AM symbiosis for plant nutrient uptake.

1. Introduction

Mineral nutrition can be a challenge for plants, in particular for non-
crop species developing in chemically-poor soils (Schjoerring et al., 2019). Fungi contribute to plant mineral nutrition, notably through
symbiotic associations, i.e. mutually beneficial associations including
bidirectional nutrient exchange (mutualism) and the formation of
complex and/or differentiated cellular structures, reflecting a level of
co-evolution between both partners. The most documented one being
the arbuscular mycorrhizal (AM) symbiosis (Wang and Qiu, 2006). It is a
widespread mutualistic symbiosis occurring between 72% of land plants
and Glomeromycotina fungi (Brundrett and Tedersoo, 2018). This
symbiosis has a major role in host plant nutrition, since Glomer-
omycotina can provide up to 100% of host plant total phosphorus (P)
(Smith et al., 2003, 2004) and up to around 20% of plant total nitrogen
(N) (Sun et al., 2022; Leigh et al., 2009). The AM symbiosis has a wide
biogeographic distribution, being found on each continent (Stürmer
et al., 2018). It is of great evolutionary relevance as it is thought to have
been a prerequisite for plant terrestrialization (Redecker et al., 2000;
Delaux et al., 2015). However, root colonization by AM fungi is known
to decrease with elevation (Kotilínek et al., 2017; Zhang et al., 2021) and
diversity of AM fungi is low in nutrient-limited soils found in alpine and
arctic environments (Geml, 2017; Kivlin et al., 2017; Stürmer et al.,
2018; Delavaux et al., 2019; Ma et al., 2023). Accordingly, these habitats
display a lower proportion of mycorrhizal plant species and a higher
proportion of so-called "non-mycorrhizal" plants unable to form any
kind of known mycorrhizal symbiosis, e.g. Brassicaceae, Cyperaceae

(Werner et al., 2018), yet still thriving in these environments (Delavaux et al., 2019, 2021).

Although most research on plant-fungus nutritional exchanges has focused on the AM symbiosis, Glomeromycotina are not the only fungi promoting plant nutrition by direct nutrient transfer. Phylogenetically diverse non-Glomeromycotina fungi have similar functions, engaging in other mycorrhizal symbioses, such as ectomycorrhizae (EcM), ericoid mycorrhizae (ErM) or orchid mycorrhizae (Brundrett and Tedersoo, 2018). EcM, first described in 1885 (Trappe, 2005), are characterised by the development of a hyphal mantle around the roots and a Hartig net in between cortical cells, whereas ErM and orchid mycorrhizae are distinguished by the formation of coils inside rhizodermal or cortical root cells. Interestingly, recent studies have also evidenced the importance of root fungal endophytes, i.e. fungi living inside host tissues during at least a period of their life, without causing morphological changes or damage to their host (Stone et al., 2000). These fungi are not considered symbiotic (sensu stricto) as they do not always form specific cellular structures inside plant roots, yet they can play comparable functional roles by transferring nutrients to their hosts (Shahollari et al., 2005; Behie et al., 2012; Hiruma et al., 2016; Almario et al., 2017, 2022; Hill et al., 2019; Hoysted et al., 2021).

Helotiales, a largely understudied fungal order, is emerging as a key
lineage in fungus-mediated nutrient acquisition by plants. These fungi
can form mycorrhizae with plants from the Ericaceae family (ErM), to
which they transfer nutrients (mainly N), while others form EcM.
Finally, some isolates have been recently shown to behave as beneficial
root-endophytes in non-ericaceous plants (Fehrer et al., 2019), even

<footnote>* Corresponding author. UMR CNRS 5557 Ecologie Microbienne, 43 bd du 11 novembre 1918, F-69622, Villeurbanne, France.
E-mail address: juliana.almario@univ-lyon1.fr (J. Almario).</footnote>
promoting plant nutrition via the transfer of nutrients in some cases (Almario et al., 2017; Hill et al., 2019). Furthermore, several root-microbiome analyses indicate that Helotiales fungi are predom-inant in the roots of plants growing in alpine and arctic environments with low nutrient availability (Toju et al., 2016; Almario et al., 2017; Jumpponen et al., 2017; Botnen et al., 2020), suggesting a key role of these fungi in the colonization success of their hosts. Even though the exact functional roles of symbiotic and endophytic Helotiales remain largely unknown, recent research has established that these fungi could be of great significance for host plant nutrition, prompting this review.

Here, we explore current knowledge about the diversity and phylogeny of plant-beneficial Helotiales fungi, we cover recent advances on how they can improve plant nutrition, finally highlighting their potential as a functional and evolutionary alternative to AM fungi for improving nutrient acquisition by land plants.

## 2. Diversity, phylogeny and distribution of plant-beneficial Helotiales

Helotiales is the largest order of Ascomycota fungi and the most diverse in terms of taxa, containing an estimated number of 5148 species distributed in 36 families and 476 genera, 144 of which are of uncertain phylogenetic placement (incertae sedis) (Wijayawardene et al., 2022). Helotialean fungi are found in various environments, e.g. water, soils and plants. However, for 70% of Helotiales species (known or hypoth-esized) their lifestyle and/or ecology remain cryptic (FUNGuild data) (Nguyen et al., 2016). The remaining 30% include mostly taxa described as saprotrophs (e.g. Chalara longpipes) or pathogens (e.g. Sclerotinia sclerotiorum, Botrytis cinerea). Interestingly, this order also includes fungi with plant-beneficial activities including ErM and EcM strains, as well as root endophytic strains promoting plant growth and/or nutrition (Acuña-Rodríguez et al., 2020; Vohník, 2020). This order includes many Dark Septate Endophytes (DSE), a paraphyletic group of root-colonizing fungi characterized by their melanized (dark) hyphae, but with still cryptic functional roles within plant tissues (Ruotsalainen et al., 2022). Indeed, more than half of all well-described DSE isolates are Helotiales (Grünig et al., 2011; Berthelot et al., 2019).

The phylogeny of known plant-beneficial Helotiales was assessed in this work using ITS sequences from strains reported to have a positive effect on plant nutrition (Supplementary file 1) as well as reference strains from other phylogenetic studies (Fig. 1) including different life-styles (Supplementary file 2). Plant-beneficial Helotiales belong to three different phylogenetic groups described within the order, with most of them found in the Hyaloscyphoid and Mollisoid taxonomic clades. Among the Helotiales lineages with defined primary lifestyles in the FUNGuild database (Nguyen et al., 2016), plant-beneficial Helotiales constitute only 12% of them, while saprotrophic and plant-pathogenic lineages account for 26% and 22%, respectively.

Strains in the Hyaloscyphoid clade include mainly ErM-forming Helotiales (Pezoloma ericae LivJKC02, EriJKE06, Rhizoscyphus ericae UAMH 6735, Hyaloscypha hepaticicola UAMH 7357, Meliniomyces variabilis T, Meliniomyces bicolor E, Cairneyella variabilis VPRI 42388) with closely related strains capable of promoting the growth of non-Ericaceae plants. For example, strains from the ErM lineage *P. ericae* (syn. *H. hepaticicola*) were shown to promote the growth and nutrition of liverwort *Cephalozia bicuspidata* (Kowal et al., 2018), which co-occurs in nature with Ericaceae plants (Upson et al., 2007). This clade also includes EcM/ErM *M. bicolor* E which associates with *Pinus sylvestris/Vaccinium vitis-idaea*, and EcM *Cadophora finlandica* PRF15 (syn. *Hyaloscypha finlandica*) which associates with *Salix caprea* (Grelet et al., 2009a; De Maria et al., 2011). Interestingly, strains closely related to these EcM lineages such as *Chloridium paucisporum* strain CBS 445.86 and *Phialophora finlandica* strain CBS 444.86 16 S (Alberton et al., 2010) promote the growth of pines in boreal forests, without forming EcM (Alberton et al., 2010).

Similarly, the Pezizelloid clade includes ErM species *Oidiodendron*

maius Zn, OM19, OMA B, UBCS18, UBCS4 and *Gamarada debralockiae* T6G9 T, and other root-beneficial fungi promoting growth of Ericaceae (Lachnum sp. ECHh) and non-Ericaceae hosts (*Cryptosporiosis rhizophila* strain CBS 109839, Hyaloscyphaceae sp. isolate 1M05). This data suggests that the ErM lifestyle has arisen at least twice in the Helotiales order, once within the Pezizelloid and once within the Hyaloscyphoid clade. Because these clades encompass ErM fungi and EcM fungi as well as beneficial root-endophytes, we hypothesize that they share similar mechanisms of plant growth promotion, regardless of their specific lifestyle, symbiotic or not. Curiously, the Pezizelloid clade included *Calycina* fungi (*Calycina alstrupii* isolate Pz162, *Calycina* as *Bisorella* PMI 857) colonizing lichens. Although their role in these associations is not documented, this suggests that the ability to associate with photosynthetic organisms is an ancient trait in this clade.

Finally, the Mollisoid clade does not include ErM or EcM strains (except for *Acephala macrosclerotium* EW76-UTF0540) but encompasses most beneficial Helotiales root endophytes described including two strains known to transfer nutrients to their hosts in a mycorrhizal-like manner (Almario et al., 2022), Helotiales sp. F229 (Almario et al., 2017) and Ascomycota sp. Da MaP I9 *Tapesia* sp. (Hill et al., 2019). Interestingly, seven of these strains, including the mycorrhizal-like fungus Helotiales sp. F229, appear to be closely related to leaf pathogens from the *Marssonina* and *Diplocarpon* genera, suggesting a major lifestyle switch in their common ancestor. The 11 other plant-beneficial strains were related to the *Phialocephala fortinii-Acephala applanata* species Complex (PAC) known to have plant growth-promoting abilities in various plants (*Pinus sylvestris*, *Carex*, *Asparagus*, *Rhododendron*), including *A. macrosclerotium* EW76-UTF0540, forming EcM structures (Vohník et al., 2005; Zijlstra et al., 2005; Grünig et al., 2006; Upson et al., 2009; Alberton et al., 2010; Surono and Narisawa, 2017, 2021; Mikheev et al., 2022). This group also includes a *Tapesia* strain (Ascomycota sp. Da MaP I9 *Tapesia* sp.) capable of transferring nutrients to its host *Deschampsia antarctica* (Hill et al., 2019).

In summary, it appears that associations between Helotiales and photosynthetic organisms (i.e. plants and lichens) have emerged several times during the evolution of this fungal order. The fact that ErM, EcM and beneficial endophytic lifestyles coexist within this order suggests a pre-disposition within these lineages toward plant-beneficial associations. The functional traits underpinning the ability of Helotiales to colonize plant roots and the known mechanisms by which they improve plant nutrition are reviewed below.

The biogeography of these potentially beneficial plant-associated Helotiales lineages is poorly described. In an effort to bridge this gap, we extracted biographical information for fungal species hypothesis (SH) matching the ITS sequences for all strains in Fig. 1 from the GlobalFungi database, which includes fungal ITS sequence data from 846 studies covering diverse samples (Větrovský et al., 2020) (Fig. S1).

In the Hyaloscyphoid clade, common patterns of distribution can be seen for ErM- and EcM-forming Helotiales and beneficial endophytes. As expected, they are detected in root/rhizosphere, soil and litter samples, but seem enriched in shoots. This surprising result is actually associated to their over-representation in moss shoots in one study (Cook et al., 2022), and might not be relevant for other plants. These plant-beneficial lineages also seem to be present in most biomes analyzed including forest, woodland, tundra, and aquatic environments, which might be associated to the wide distribution of their host plants.

In contrast, plant-beneficial Helotiales within the Mollisoid clade show a higher enrichment in root/rhizosphere samples, as expected for root endophytes. Interestingly, a part of the Mollisoid clade seems to be highly enriched in water samples. This could imply a secondary water-associated lifestyle or perhaps even a relation with aquatic plants for these fungi. This points to water as an additional reservoir of plant-beneficial Helotiales especially in aquatic, tundra and grassland biomes, where these lineages seem enriched. Moreover, the distribution of certain lineages (Ascomycota sp. Da PoT1 C7 and MaP I9 *Tapesia* sp.) also seems specific to the Antarctica desert, where they are enriched in
<figure><img src="image_3.png" /></figure>

Tree scale: 0.1

*Plant-associated lifestyles

Biotroph ☐ Necrotroph ☐

Ectomycorrhizal ☐ Ericoid mycorrhizal

Endophyte ☐

Beneficial endophyte

(caption on next page)
**Fig. 1. ITS-based phylogenetic tree of Helotiales strains linked to improved plant nutrition.** ITS sequences from Helotiales strains with a described positive effect on plant nutrition were extracted from NCBI. Sequences from strains used by Johnston et al. (2019) or by Li et al. (2021) to establish Leotiomycetes phylogeny on entire genomes as well as sequences from known lichen-associated Helotiales from Diederich et al. (2018) and Suija et al. (2015), were also included (shown in bold; “T” indicates type strains). Helotiales phylogenetic clades described by Johnston et al. (2019) are indicated. Sequences were aligned using Mafft (L-INS-i method with default parameters and “leave gappy regions” option). Residuals to construct the tree were selected using Gblocks (with relaxed parameters). The tree was constructed on 363 residues (PhyML, GTR model, SH-aLRT with 1000 repetitions, in Seaview). Sequences from non-Helotiales Leotiomycetes: *Pseudogymnoascus destructans* (Thelebolales), *Thelebolus stercoreus* (Thelebolales), *Naemacyclus fimbriatus* (Marthamycetales), were used for rooting. The scale represents the number of differences between sequences. When available, the fungal lifestyle was indicated, based on previous studies (Supplementary file 2). Plant pathogens are described either as biotrophs (i.e. colonize living plant tissue and obtain nutrients from living host cells) or necrotrophs (i.e. colonize living plant tissue and obtain nutrients from dead tissues). Symbiotic Helotiales are marked as either forming ericoid mycorrhiza or ectomycorrhiza, and root endophytes are either characterized as endophytes with no known effect on plants or as beneficial root endophytes. Purple NT symbols indicate strains shown to transfer nutrients to their hosts.

plant shoot samples.

Plant-beneficial Helotiales from the Pezizelloid clade have a more stochastic distribution except for *Oidiodendron maius*, which is enriched in root/rhizosphere and wetland samples, rather than in shrublands where Ericaceae are usually abundant.

These observations show that plant-beneficial Helotiales exhibit varied biogeographical patterns, particularly favouring cold and humid environments. However, these results should be interpreted with caution due to the sparsity of the data, and biases when attributing SH identities to studied strains based on partial ITS sequences.

## 3. How do Helotiales improve plant nutrition?

Several experiments have shown that inoculation with Helotiales strains can promote plant growth, leading to higher biomass, and higher nutrient levels (P or N) under laboratory conditions (Newsham, 2011; Berthelot et al., 2019; Malicka et al., 2022). These effects are reported for ErM and EcM Helotiales (Smith and Read, 2008; Wei et al., 2022), but also root endophytic Helotiales engaging in non-symbiotic associations with plants (i.e. associations not entailing the formation of specialized structures or organs within plant roots, and for which mutual benefits are not always proven) (meta-analyses Newsham, 2011; Mayerhofer et al., 2013). We hypothesize that Helotiales' plant growth-promotion effects mainly come from their enhanced capacity to mineralize organic matter and transfer the mined nutrients to their host plants.

### 3.1. Organic matter mineralization

Plants acquire nutrients in mineral forms from the soil, and very rarely from organic sources (Paungfoo-Lonhienne et al., 2008). However, soil nutrient contents directly available for plants are very low. For example, soluble mineral phosphate, the only P source directly available for plants, represents 1% of all soil P (Richardson and Simpson, 2011). Most of soil P and N is contained in organic matter, and these nutrient sources are not readily accessible to plants. However, organic matter decomposition releases nutrients such as P or N into the soil solution via mineralization and solubilisation processes, thereby increasing nutrients' bioavailability for plant use. Bacterial and fungal microorganisms are major decomposers (Setälä and McLean, 2004), and amongst fungi, the Helotiales appear to be particularly efficient at breaking down complex organic matter (Martino et al., 2018; Guerreiro et al., 2023).

In addition to their symbiotic traits, ErM-forming Helotiales have retained a saprotrophic lifestyle as they can live independently from their ericaceous hosts (Knapp et al., 2018; Martino et al., 2018; Mesny et al., 2021), obtaining nutrients from organic matter thanks to their enzymatic repertoires. In a symbiotic context, these same traits allow ErM Helotiales to mobilize organic N and P forms in the rhizosphere, making them available for the plant. These fungi release extracellular enzymes, which degrade N and P organic forms such as proteins-polyphenols complexes (sources of mineral N and P) (Leake and Read, 1990a; Bending and Read, 1996), protein polymers, chitin (N) (Bajwa et al., 1985; Leake and Read, 1990b, 1991), phytate, nucleic acids (P, N) (Lemoine et al., 1992; Leake and Miles, 1996; Myers and

Leake, 1996a; Chen et al., 1999) (reviewed by Smith and Read, 2008). They can also degrade complex C sources (cellulose, pectin, polyphenols), which can help release co-precipitated N compounds (Jalal et al., 1982; Jalal and Read, 1983).

In mycorrhized Ericaceae, fungal mineralization activities promote plant nutrition in presence of organic N and P. In planta experiments with the Ericaceae *Vaccinium corymbosum* and *Gaultheria shallon* inoculated with their respective ErM symbionts *H. hepaticicola* (*R. ericae* UAMH 6735) and *O. maius* (*O. maius* UBSC4, UBSC18), showed an increase in plant biomass (120–300%) and N content (~130–160%) when a source of organic N was added to the medium as peptides (tripeptide glutathione or alanine units of 1–6 amino acid residues) or purified BSA (Bovine Serum Albumin protein) (Bajwa and Read, 1985; Bajwa et al., 1985; Xiao and Berch, 1999). In experiments with more complex organic constituents, such as plant litter or fungal cell walls, similar increases in plant biomass were observed (200–500%) on *H. hepaticicola*-inoculated (i.e. *R. ericae* UAMH 6735) *Vaccinium macrocarpon* and *C. vulgaris* plants (Kerley and Read, 1995, 1998). This could hold great significance, as chitin – the primary component of fungal cell walls – can be the main soil N pool in *C. vulgaris*-dominated heathlands (Kerley and Read, 1997). As described for N, ErM Helotiales can also enhance P nutrition of Ericaceae via similar mechanisms. The most abundant sources of organic P in soil are monophosphoesters notably phytates, and diphosphoesters such as nucleic acids (Bieleski, 1973). *H. hepaticicola* (*R. ericae* UAMH 6735) can decompose purified and unpurified nucleic acids, enhancing P content of its host *V. macrocarpon* by 400% compared with uninoculated plants (Leake and Miles, 1996; Myers and Leake, 1996b). However, even though ErM Helotiales are able to assimilate P from phytates (Gibson and Mitchell, 2005), to the best of our knowledge, no study has yet shown direct beneficial effects of phytate mineralization by ErM Helotiales, on Ericaceae P nutrition. These observations suggest that mineralization of P and N sources by ErM Helotiales contribute to the adaptation of Ericaceae to environments with high amounts of poorly-decomposed organic matter (Adamczyk et al., 2016).

Helotialean root endophytes can also exploit organic matter degradation thanks to their enzymatic repertoires, with similar beneficial effects on plant nutrition. Indeed, in a meta-analysis across 18 studies, Newsham (2011) showed that the inoculation of root endophytic Helotiales fungi tended to increase shoot N content by 113% compared with the uninoculated control. In pure culture, the root endophytes *H. finlandica* (*Phialophora finlandia*) and *P. fortinii* can degrade proteins and nucleic acids as N and P sources (Caldwell et al., 2000), whereas *P. fortinii* (*P. fortinii* pkc10, *P. fortinii* NoF 3140) uses casamino acids and urea as N sources (Surono and Narisawa, 2017) or phytates as P sources (Mikheev et al., 2022). Moreover, several endophytic strains belonging to *P. fortinii* (strain pkc10), *Leptodontidium orchidicola* (strain UAMH 8151), *Lachnum* (strain EcHh), or *Cadophora* (strains Fe06, Ed6d, DSE1049) as well as other non-identified Helotialean strains can also degrade C-polymers (cellulose, cellobiose, starch) derived from plants under *in vitro* conditions (Caldwell et al., 2000; Bizabani and Dames, 2015; Berthelot et al., 2016; Knapp and Kovács, 2016; Surono and Narisawa, 2021). These activities are similar to the ones found for ErM-forming Helotiales, pointing towards mineralization as a common trait of plant beneficial Helotiales. The question of whether
non-symbiotic root endophytic Helotiales could have similar effects on plant nutrition as ErM-forming Helotiales has been addressed by a few studies in planta. Upson et al. (2009) inoculated three Heliotalean fungi isolated from *D. antarctica* roots and belonging to *Oculimacula yallundae* (Ascomycota sp. Da Bac I4) or *Tapesia* (Ascomycota sp. Da Bac I9, C7) lineages. This resulted in an increase in plant shoot biomass (by 51–178%), P content (by 47–65%), and N content (by 52–123%) in presence of organic N (casein hydrolysate), while in presence of an inorganic N source (ammonium sulfate) the fungi had no effect on plant biomass, and even reduced N and P contents (Upson et al., 2009). In a similar way, in presence of organic N and P sources, *P. fortinii* (*P. fortinii* pkc10, Wang and Wilcox SE24) improves the growth of several host plants including *Asparagus officinalis* (Surono and Narisawa, 2017), *Brassica oleracea* var. *Capitata*, *Spinacia oleracea* (Surono and Narisawa, 2021) and *Pinus contorta* (Jumpponen et al., 1998), with biomass increases ranging from 50 to 303% compared with uninoculated plants. In the case of P, other strains belonging to the PAC increased the pool of bioavailable P in the soil solution by 150% compared with uninoculated substrate (Della Monica et al., 2015). Interestingly, in the same experiment, the AM fungus *Gigaspora rosea* did not enhance the pool of bioavailable P suggesting that Helotiales root endophytes could be more effective at mineralizing or mobilizing organic matter than AM fungi. Furthermore, co-inoculation of AM fungi with root endophytic Helotiales *P. turiciensis* or *P. piceae* dramatically increased plant P content (by 400%), which suggests a strong potential synergy between these two groups of fungi. In this model, Heliotalean fungi could mineralize organic P and provide AM fungi with inorganic P that they can transfer directly to clover (Della Monica et al., 2015). All these studies provide evidence that mineralization activities of root endophytic Helotiales could play an important ecological role for plant nutrition, especially in environments with poorly-decomposed organic matter. The functional role of these Helotiales could be similar to what has been described for non-Heliotalean EcM fungi capable of mobilizing organic P sources via the secretion of enzymes, thereby mediating the adaptation of the host to soils poor in inorganic P (Meeds et al., 2021).

The potential of Helotiales fungi to mineralize organic matter is also documented at the genetic level. Comparative genomic studies point that Helotiales, both ErM and root endophytes (*P. subalpina* UAMH11012 and *Cadophora* sp. strain DSE1049) display a large apparatus for organic matter degradation including expanded repertoires of CAZymes (Carbohydrate Active Enzymes), lipases and proteases, with a great potential to release mineral forms of N and P and/or free co-precipitated N and P compounds (Knapp et al., 2018; Martino et al., 2018). This contrasts with (i) AM fungi, which display a very small CAZyme repertoire and loss of genes coding for lignocellulose degradation (Morin et al., 2019), and (ii) EcM fungi showing a similar contraction in their CAZyme arsenal (Miyauchi et al., 2020). Large CAZyme repertoires are often associated to fungal saprotrophic life-styles, yet the larger ranges of CAZymes are observed in the genomes of ErM and root endophytic Helotiales, suggesting that CAZymes and specifically cell wall degrading enzymes might be key to the interactions between Helotiales and plants (Kohler et al., 2015; Knapp et al., 2018; Martino et al., 2018; Miyauchi et al., 2020; Mesny et al., 2021; Melie et al., 2023). For example, Martino et al. (2018) inoculated four ErM-forming Helotiales (*R. ericae* UAMH7375, *M. variabilis* F, *M. bicolor* E, *O. maius* Zn) on *Vaccinium myrtillus* plants, which resulted in higher expression of fungal genes coding for secreted enzymes degrading pectin and hemicellulose, proteases (aspartic proteases or glutamic proteases) and lipases. It remains however to be determined if these genes are only involved in the intraradical establishment of Helotiales in roots, or if they could also mediate N and P release from organic matter in extra-radical hyphae. These results suggest that mineralization of organic matter is one of the main mechanisms by which ErM-forming Helotiales and root endophytic Helotiales improve plant nutrition.

## 3.2. Direct nutrient transfer

Fungus-mediated nutrient transfer from soil to plant roots is best described for AM fungi and entails several steps. It starts with fungus taking up nutrients like N and P via their extraradical hyphae, which can be facilitated by the prior solubilisation and mineralization of unavailable nutrient forms. The absorbed nutrients are then transported over long distances along the mycelium network —more than 14 cm for *Glomus* interacting with maize roots (Jansa et al., 2003)— in the form of polyphosphate for P (Ezawa et al., 2001), and glutamine and arginine for N (Govindarajulu et al., 2005). Once they reach the intraradical hyphae they are translocated from the fungal to the plant cell via arbuscules and/or other fungal structures developed inside the apoplastic space of plant root cells. This nutrient transfer is thought to be active and has also been described for K, Fe and other micronutrients (Wang et al., 2017; and references within).

Similar processes have been described for direct nutrient transfer during ErM symbioses between Helotiales fungi (genera *Hyaloscypha*, *Gamarada* and *Cairneyella* and the species *O. maius*) and Ericaceae plants, but studies remain scarce. Indeed, ErM-forming Helotiales take up soil nutrients via their extraradical hyphae, e.g. N as ammonium, nitrate, or even amino acids, which are then transferred to the host plant (Bajwa and Read, 1986; Kosola et al., 2007). However, little is known about all the P forms absorbed by the fungus but long distance P transport seems to rely on intra-cellular polyphosphates (Straker and Mitchell, 1985). These nutrients are then transferred to intraradical hyphae forming the characteristic ErM structures called “coils” inside root cells. They are fine compact intracellular hyphal structures in rhizodermal cells of healthy ericoid root hairs (Read, 1996), where nutrient exchanges are thought to take place. Upon reaching this structure, nutrients are believed to be either actively translocated to plant cells or passively released as observed for orchid mycorrhizae (Smith and Read, 2008; Bougoure et al., 2014). Comparative genomic studies show that in ErM-forming Helotiales, gene families coding for proteins involved in nutrient uptake/exchange (MFS for major facilitator superfamily and sugar transporters) are expanded compared with those in saprotrophs and pathogens (Martino et al., 2018). Moreover, fungal-mediated amino acid uptake was shown via single cell electrophysiology in a study on *C. vulgaris* (Sokolovski et al., 2002). Root cortex cells colonised with *H. hepaticicola* (*Hymenoscyphus ericae* (Read) Korf and Kernan, UAMH 6735) showed up to four times larger depolarizations than non-colonized cells when amino acids were added to the growth media of the inoculated plant, which indicates an increase in amino acids influx in fungal-colonised cells. These results support the concept of an active translocation of nutrients from the fungus to the plant during ErM symbiosis.

Among the four taxonomic groups of ErM-forming Helotiales, direct nutrient transfer has only been proven for the *H. hepaticicola* clade. Using radiolabelling, it was shown that a strain of *H. hepaticicola* (strain UAMH 7357) is capable of transferring $ ^{32}P $ (as orthophosphate) to its Ericaceae hosts *Calluna vulgaris* and *Vaccinium oxycoccus* (Pearson and Read, 1973), while other strains (*P. ericae* AC2, AC21) were shown to transfer $ ^{15}N $ as glutamine or ammonium to *V. macrocarpon* and *V. vitis-idaea* (Grelet et al., 2009b). Moreover, inoculation of another strain of *H. hepaticicola* (*R. ericae* UAMH 6735) on *V. macrocarpon* under N limiting conditions also modified plant N absorption kinetics, increasing the $ ^{15}NO_3^- $ influx eightfold compared with the non-inoculated control, indicating a promotion of plant N acquisition especially in conditions with little N directly available for the host plant (Kosola et al., 2007). Carbon transfer from the host to the ErM Helotiales species *H. hepaticicola* (strain UAMH 7357) was also evidenced based on fungal accumulation of $ ^{14}C $ carbohydrates in $ ^{14}CO_2$-fed plants (Pearson and Read, 1973; Stribley and Read, 1974), suggesting a reciprocal trade of nutrients. Similarly, in the ErM association between *V. vitis-idaea* and *M. variabilis* F and *M. bicolor* E isolates, the extraradical fungal mycelium was enriched in $ ^{13}C $ (by 10 atom%) and leaves of $ ^{13}C$-labelled inoculated
plants were significantly enriched in ¹⁵N (by 2 atom %), in comparison with non-mycorrhizal controls (Grelet et al., 2009a).

For the three other ErM Helotiales taxa (i.e. *O. maius*, *G. debralockiae*, *C. variabilis*), no data is available on direct nutrient transfers and these fungi are actually classified as ericoid mycorrhizal fungi solely based on morphological evidence of coil formation in Ericaceae root cells. However, plant growth-promotion effects have been found with *O. maius* based on enhanced plant growth, and shoot N and P content of *Rhodo-dendron*, under laboratory conditions (Vohník et al., 2005; Wei et al., 2016a, 2016b, 2020, 2022). Similarly, *C. variabilis* MG60 promoted the growth of *Woollsia pugens* (Palmer et al., 2007). Although evidence of direct nutrient transfer by ErM Helotiales is very limited and stems mainly from *in vitro* experiments, ericoid mycorrhizae have been proposed to play major roles in C and N biogeochemical cycles and plant nutrition in forests and subalpine environments (Ward et al., 2022). Moreover, most of our knowledge on the ErM symbiosis comes from experiments with *H. hepaticicola* strains and plant species growing in heathlands with acidic soils, while Ericaceae are found in a wider range of environments where other ErM fungal lineages might drive these exchanges. Thus, broader studies of the diversity ErM-forming fungi (Leopold, 2016; Vohník, 2020), are needed to fully understand Ericaceae mineral nutrition and their adaptations (Vohník and Réblová, 2023).

In addition to ErM-forming strains, other Helotiales taxa have been found as root endophytes in non-Ericaceae plants (Zijlstra et al., 2005; Newsham, 2011; Walker et al., 2011; Maciá-Vicente et al., 2020). These fungi may display plant growth-promoting activities, including direct nutrient transfer previously thought to be restricted to classical mycorrhizal fungi (including ErM and AM taxa) (van der Heijden et al., 2015; Almario et al., 2017; Hill et al., 2019). These Helotiales are considered "mycorrhizal-like" fungi as they may transfer P or N to their hosts without forming any specialized structures, such as coils or arbuscules (Almario et al., 2017, 2022; Kowal et al., 2018; Hill et al., 2019). These associations are thought to be functionally analogous to a mycorrhizal symbiosis. For instance, Helotiales sp. strain F229 retrieved from the roots of the Brassicaceae (non-mycorrhizal) *Arabis alpina* growing in a low P alpine soil, promoted plant growth (by 52%) and P acquisition (by 61%) (Almario et al., 2017). Using ³³P radiolabelled orthophosphate, Almario et al. (2017) further showed that this root endophyte was capable of transferring P to its host *A. alpina* independently of the concentration of P present in the medium, which means that this process is not controlled by the plant P status. Similarly, a Helotiales DSE isolate from the genus *Tapesia* (referred to as Ascomycota sp. Da MaP I9 *Tapesia* sp.), retrieved from the roots of mycorrhizal *D. antarctica* (*Poaceae*) growing on a subantarctic island, was able to transfer N to its host (Hill et al., 2019). NanoSIMs imaging showed that ¹⁵N was transported as D- and L-trialanine to *D. antarctica* roots after their exposition to the ¹⁵N source for 5 min and ¹⁵N was recovered in the intercellular space between the root cortical cells of *D. antarctica* only when inoculated with the *Tapesia* strain (Hill et al., 2019). Additionally, individual root cells of plants supplied with D- or L-trialanine were more enriched with ¹⁵N when colonised with the Helotiales endophyte than in non-colonised controls. However, carbon flux was not measured in any of these interactions, so it is not known if transfer of C from the plant to these fungal endophytes takes place. In addition, whether these partners are involved in a cooperative or mutualistic interaction remains an open question, even though the term "symbiotic" is often used (Sarkar et al., 2021). Similar mycorrhiza-like associations are found in non-vascular plants, e.g. between the liverwort *Cephalozia bicuspidata* and the Helotiales *P. ericae* (*P. ericae* LivJKC02, EriJKE06) isolated from this non-mycorrhizal plant species (Duckett and Read, 1995; Kowal et al., 2018). For this particular interaction, the transfer of ³³P from the fungus to the plant was shown to be associated with ¹⁴C transfer from the plant to the fungus (using ¹⁴CO₂ labelling), with an allocation of 0.27% of the total C fixed by the plant during the labelling period of 5 h. This fungus-to-plant nutrient transfer was proposed to explain the doubling

of moss biomass upon fungal inoculation. We propose that in arctic-alpine environments, where AM fungi are thought to be less relevant, these mycorrhizal-like Helotiales could be essential for the nutrition of non-Ericaceae plants. Key points to address this hypothesis will be the quantification of these transfers in the natural soil environment, with natural plant and natural fungal populations, and the phylogenetic characterisation of these mycorrhizal-like Helotiales. In the case of EcM Helotiales, we found no study showing fungus-to-plant nutrient transfer.

Taken together, these studies suggest that the ability to transfer nutrients to plants is a common trait among plant-beneficial Helotiales. Interestingly, these transfers do not necessarily entail the formation of complex plant-fungus cellular structures as they are observed both in ErM symbioses, characterized by the accommodation of coil-like fungal structures inside plant cells, and mycorrhiza-like associations, with more limited intra-cellular fungal colonization (Fig. 2). Whether these mycorrhiza-like associations can be simply considered as a new type of mycorrhizal symbiosis is a subject of debate and depends rather on the definitions considered. From our perspective, a true mycorrhizal symbiosis would entail a proven bidirectional nutrient exchange (mutualism) and the formation of complex and/or differentiated cellular structures, reflecting a level of co-evolution between both partners. Furthermore, biogeographical studies are still needed to establish the spread and specificity of these associations.

### 4. Mycorrhizal-like Helotiales as an alternative to the AM symbiosis in nutrient-limited environments?

Helotialean symbiotic and non-symbiotic interactions are of particular importance for plant nutrition in environments where the AM symbiosis is absent. This situation can occur in environments with very low soil fertility (Lambers and Teste, 2013; Ma et al., 2023), where AM fungi are not thriving and their diversity is low (Geml, 2017; Stürmer et al., 2018), such as arctic and alpine environments (Brundrett and Tedersoo, 2018). These soils are often very limiting in available N and P, especially on acidic crystalline bedrocks (Bonneau, 2005). Interestingly, these habitats display a diversified range of non-mycorrhizal plant species, i.e. plant lineages having lost the genetic ability to form a functional AM symbiosis (such as the Brassicaceae), and they represent a higher proportion of the plant community than in other climatic conditions (Read and Haselwandter, 1981; Chapin and Körner, 1995; Delavaux et al., 2019, 2021). We suggest that in these environments, Helotiales might play key roles sustaining the nutrition of mycorrhizal Ericaceae (via ErM symbiosis establishment) and non-mycorrhizal plants (via non-symbiotic associations with root endophytic Helotiales).

In contrary to the ancestral AM symbiosis (450 Mya), the ErM symbiosis appeared more recently in plant evolution (100–110 Mya; Schwery et al., 2015) and seems to have emerged from an Ericaceae ancestor having lost the capacity to form a functional AM symbiosis, subsequently developing the ability to associate with ancestral Helotiales fungi. ErM biogeographical studies show that plants establishing this symbiosis are found on every continent but Antarctica, and are highly prevalent in alpine biomes and high tropical mountains. Their relative abundance increases towards the poles, and they represent a major part of the vegetation in boreal forest and tundra biomes (Kohout, 2017 and references within). These environments are characterised by acidic soils, low temperature and slow degradation of organic matter, which results in low-nutrient availability for plants (Kerley and Read, 1998). The ErM symbiosis may have arisen as an evolutionary adaptation of Ericaceae ancestors to mobilize nutrients from organic matter (Kerley and Read, 1998) by partnering with Helotiales, more efficient than AM fungi to degrade organic matter, and capable of both mineralizing and transferring nutrients to plants. ErM-forming Helotiales are crucial to the nutrition of Ericaceae, and interestingly other non-ErM Helotiales taxa are commonly found in roots of non-Ericaceae hosts in alpine environments (Zijlstra et al., 2005; Newsham, 2011; Glynou
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Schematic representation of beneficial plant-Helotiales associations involving fungus-to-plant nutrient transfers and nutrient mobilisation in the soil. Selected EcM (De Maria et al., 2011) and ErM (Pearson and Read, 1973; Kosola et al., 2007; Grelet et al., 2009a), as well as mycorrhiza-like associations (Almario et al., 2017; Kowal et al., 2018; Hill et al., 2019) are shown with described patterns of fungal colonization and nutrients transferred. The background illustrates the environments where these associations are thought to preferentially occur. The complete list of the supporting publications is given as supplementary data (Supplementary file 1).</figcaption></figure>

et al., 2016; Jumpponen et al., 2017; Toju et al., 2019; Botnen et al., 2020; Maciá-Vicente et al., 2020).

Several root-microbiota analyses have shown a great prevalence of Helotiales fungi in arctic and alpine environments (Glynou et al., 2016; Toju et al., 2016; Almario et al., 2017; Jumpponen et al., 2017; Botnen et al., 2020; Maciá-Vicente et al., 2020), suggesting they might have a still unknown ecological role in these habitats. Moreover, Helotiales are abundant in roots or rhizoids of non-mycorrhizal plants such as some lineages of non-vascular plants like Jungermanniales (liverworts), mosses (Zhang et al., 2013; Chen et al., 2022), and angiosperms such as Brassicaceae (e.g. *Arabis alpina*, *Microthlaspi* spp.; Almario et al., 2017; Macia-Vicente et al., 2020), Caryophyllaceae (e.g. *Silene* spp.; Toju et al., 2019; Botnen et al., 2020) and Cyperaceae (e.g. *Carex* spp.; Gao and Yang, 2016; Toju et al., 2019; Botnen et al., 2020; Liu et al., 2023). Given the similar behaviour and genomic traits these endophytes share with ErM fungi, we propose they could sustain the growth and nutrition of non-mycorrhizal plants by providing access to nutrients contained in organic matter via mineralization and direct nutrient transfer. We hypothesize that these interactions are actually mediating the adaptation of non-mycorrhizal plants to low nutrient arctic-alpine soils. Because non-mycorrhizal lineages appeared relatively recently (30 million years ago; Werner et al., 2018), their associations with these root endophytic Helotiales might be at a 'waiting room' stage, predisposing the partners to an evolution towards a future symbiotic mycorrhizal association (Selosse et al., 2009; van der Heijden et al., 2015; Strullu-Derrien et al., 2018).

**5. Conclusion and outlook**

Fungi from the Helotiales can improve plant nutrition during symbiotic and non-symbiotic associations, by mineralizing and transferring nutrients to their hosts. Research from the last 10 years has brought up that these nutritional effects are not limited to ErM symbiotic Ericaceae plants as it was thought, but that non-symbiotic root endophytic

Helotiales can directly transfer mineral nutrients to other plants like non-mycorrhizal Brassicaceae. Here, we show that these mycorrhizal-like Helotiales belong to different lineages and are phylogenetically distinct from ErM Helotiales, which suggests that the capacity to transfer nutrients to plants is widespread in three clades from this order. As these fungi seem to be highly prevalent in soils with poor nutrient availability, such as in arctic-alpine soils, it is possible that they actually mediate plant adaptation by sustaining plant nutrition in these environments. These associations could be of extreme importance particularly for non-mycorrhizal arctic-alpine plants from the Brassicaceae, Cyperaceae and Caryophyllaceae families, unable to rely on the ancestral AM symbiosis to ensure their nutrition.

Despite recent breakthroughs, the ecology and significance of Helotiales fungi remain poorly understood. Further research is necessary in three directions. First, the diversity of plant-beneficial Helotiales remains particularly ill-documented, especially in non-AM plants. Meta-barcoding provides information on the distribution of Helotiales in various plant environments, but genomic information is urgently needed to improve Helotiales phylogeny and understand the eco-evolutionary processes behind their endophytic and plant-beneficial lifestyles. Secondly, functional approaches are required to identify functions and genes associated to their plant-beneficial effects. This includes quantifying nutrient transfers to the plant and elucidating the underlying mechanisms, with the hypothesis they might be highly conserved at the order level. Finally, this order represents an interesting source for new fungal biofertilizers capable of both effectively mobilizing nutrients from the soil and transferring the scavenged nutrients to the plant. They could be therefore used to improve fertilization efficacy by increasing crop nutrient uptake.

**CRediT authorship contribution statement**

**Pauline Bruyant:** Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Yvan Moëlle-**
**Locozc:** Writing – review & editing, Supervision, Project administration, Methodology, Investigation, Conceptualization. **Juliana Almario:** Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.

Acknowledgements

Funding: This work was supported by the ANR (Agence Nationale de la Recherche) [Projet-ANR-22-CE02-0018], the French National program EC2CO (Ecosphère Continentale et Côtière) [Projet MicroPhos] and the Ministère de l'Enseignement supérieur et de la Recherche (Ph.D. fellowship for P.B.).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.soilbio.2024.109363.

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