Metabarcoding Reveals Diverse Endophytic Fungal Communities in Vaccinium Myrtillus Plant Organs and Suggests Systemic Distribution of Some Ericoid Mycorrhizal and DSE Fungi

Genome sequencing data revealed unexpected similarities among plant-interacting fungi belonging to different ecological guilds. In particular, the sequenced genomes of ericoid mycorrhizal fungi (ErMF) showed closer similarities with genomes of plant endophytes than with those of other mycorrhizal fungi. ErMF are typically associated with roots of plants in the Ericaceae, but it has never been investigated whether they also colonize other organs of their natural hosts. Here, we applied a metabarcoding approach to describe the fungal community associated with the different organs of Vaccinium myrtillus plants collected in the field. Taxa in the Helotiales and Sebacinales, known to include ErMF, characterize the root endosphere, together with Agaricales and Lecanoromycetes, while the stems were enriched in Agaricomycetes , Tremellomycetes and Pleosporales , the leaves were enriched in Sordariomycetes , Hysteriales , and the flowers were enriched in Dothideomycetes . Operational Taxonomic Units attributed to known or putative ErMF and Dark Phialocephala fortinii , were found in all the plant organs. The ErMF Oidiodendron sp. was rarely detected in organs other than roots in field samples, but we could detect its presence in the above-ground organs of V. myrtillus grown in vitro . This first report of ErMF colonizing the above-ground tissues of the host plant mirrors their evolutionary closeness with endophytes and increases the list of fungi found to occupy several niches.


Introduction
Plants live closely associated with complex microbial communities, or microbiota, that colonize the plant surfaces (e.g., rhizosphere and phyllosphere) as well as internal tissues (the endosphere), and include nematodes, fungi, unicellular eukaryotes, bacteria, archaea and viruses 1 . The plantassociated microbiota can play a key role for plant health, productivity and development, and a plant with its associated microbiota, the "holobiont", can be considered as a single entity that evolves in the environment and time, thanks to the co-evolution of the single components interacting with each other 2 .
Microorganisms inhabiting the plant internal tissues for at least part of their lifetime are termed endophytes 1 . In particular, endophytic fungi are functionally dominant in the plant microbiota; they are ubiquitous and have been found in all species of plants studied to date 3 . Endophytic fungi live in the host tissues without causing evident symptoms 4 and are often considered to be beneficial to their host plants because they may provide resistance against pathogens and insect herbivory 5 . They can also confer stress tolerance, such as salt and heat tolerance 6 and promote plant root formation and shoot growth 7 . On the other hand, endophytic fungi could become pathogens under stressful conditions, or they could have long latent periods 8 .
Rodriguez et al. 3 classified endophytic fungi according to their colonization pattern and phylogeny.
Class I endophytes, also known as clavicipitaceous endophytes, includes phylogenetically related species that form systemic intercellular infections in the shoots of some grasses, being primarily vertically transmitted. Class II, III and IV include a taxonomically and functionally highly diverse group of non-clavicipitaceous species. Class II species can extensively colonize both above-and below-ground plant tissues, can be transmitted both horizontally and vertically via seed coats, seeds or rhizomes and confer habitat-specific stress tolerance to host plants 6 . Class III species are characterized by a high diversity within a single plant, where they are limited to above-ground tissues with highly localized infections, and can be horizontally transmitted. Class IV species, namely the Dark Septate Endophytic (DSE) fungi, are restricted to the roots, which they colonize extensively in a wide range of host plants, and are horizontally transmitted. This classification of fungal endophytes excluded the mycorrhizal fungi because, in addition to internal root tissues, these fungi grow outside the rhizosphere into the soil. Mycorrhizal fungi colonize the plant root tissues, where they form intimate symbioses whose morphological and functional features depend on the plant and the fungal taxonomic position 9 . The formation of specialized fungal structures within the plant tissues also excluded mycorrhizal fungi from the definition of endophytes given by Wilson 10 "fungi or bacteria which, for all or part of their life cycle, invade the tissues of living plants and cause unapparent and asymptomatic infections entirely within plant tissues but cause no symptoms Irrespective of the definition and spectrum, many endophytic fungi seem to be unequally distributed in the different plant compartments. A similar pattern has been found in the diversity and distribution of bacteria associated with plant surfaces and internal plant tissues, both above-and below-ground, where it has been suggested that these plant compartments may represent a major selective force that shapes the composition of plant-associated microbiota 2 . When compared to communities of bacterial endophytes, that have been widely investigated, variation in the fungal communities within the different plant niches is still poorly known, and although several studies have focused on the plant-soil interface, less is known about the patterns of fungal diversity in the different plant compartments 11 . It is for example unclear whether the distribution in the plant depends on the taxonomic position of the fungus, or on specific constraints posed by the different plant compartments. To increase our knowledge on the fungal communities associated with aboveand below-ground plant tissues, and to address specific questions on the distribution of some key components of plant-associated fungi, we have investigated the diversity of the fungal community colonizing the internal tissues of different organs of Vaccinium myrtillus (Ericaceae) plants, a species known to form a specific mycorrhizal symbiosis.
Plants belonging to the Ericaceae family, encompassing 4426 species and around 129 genera 12 , represent important components of the heathland flora and some open forest communities worldwide. These geographically and climatically disparate habitats rely on soils that are usually very poor in mineral nutrients but can be enriched in aromatic compounds and potentially toxic metals, made readily available by the generally low pH 13 . The adaptation of Ericaceae to these stressful habitats has been largely attributed to the ability of their associated mycorrhizal fungi to increase the host plant fitness 14 . The role of non-mycorrhizal fungal endophytes in the adaptation of Ericaceae to stressful conditions is far less understood, although Class IV endophytes, namely the DSE fungi 3 , are commonly isolated from the roots of ericaceous plants [15][16][17][18][19] and inoculation with DSE fungi under controlled conditions enhanced plant performance 20 . Furthermore, some DSE fungi seem to have a potential to form ErM 21 .
Besides playing a crucial ecological role in heathland habitats, some genera of Ericaceae have a commercial interest as agronomic cultures in the flower and horticultural industry, both as food and nutraceutical sources, thanks to their richness in secondary metabolites 22 . Ericoid mycorrhizal fungi have been demonstrated to influence not only plant fitness in the field, but also some plant phenotypic traits, such as flower size and fruit number and quality 23  Basidiomycetes species in the genus Serendipita (Sebacinales, Agaricomycetes) are also common inhabitants of ericaceous roots, where they form typical hyphal coils 32 . A species in the order Trechisporales (Agaricomycetes), identified by Vohník and colleagues 33 from Vaccinium spp., has been considered as a putative ErMF because it forms intracellular structures with a unique morphology described as a "sheathed-ericoid" mycorrhiza.
Whereas the root-associated fungal communities of ericaceous plants have been investigated by culture-dependent and independent methods in many recent studies 16,17,19,[34][35][36][37][38] , few investigations have focused on the fungal diversity in the above-ground organs. Li and colleagues 39  Thus, we hypothesize that some root-associated fungi may be more versatile in their trophic strategies and colonization potential than traditionally thought. A further aim of this work was therefore to verify if fungi typically described as being restricted to the root endosphere of ericaceous plants, like ErMF and DSE fungi, can also colonize the above-ground plant organs.

Fungal diversity associated with the different plant organs
The fungal communities associated with the four different organs of V. myrtillus were revealed by high-throughput sequencing of the fungal ITS2 region. After removal of low-quality reads, we obtained in total 2,863,742 high quality reads (maximum counts per sample: 188,914; minimum counts per sample: 93,654) corresponding to 1,621 Operational Taxonomic Units (OTUs; 97% similarity), among which 1,186 had ≥ 2 counts. After discarding OTUs with low counts (less than 10 reads) and low standard deviation (see material and methods), 749 OTUs were retained.
The alpha diversity of fungal communities in the four different plant organs analyzed (i.e., roots, stems, leaves and flowers) was assessed by calculating the Chao1 and Shannon indices. The Chao1 index, which estimates richness based on taxa abundance, showed no significant differences among organs (Kruskal-Wallis p-val=0.08; Supplementary Fig. S1), while the Shannon index, that considers both richness and evenness (abundance distribution across species), revealed a significant At the phylum level ( Supplementary Fig. S2), the fungal population associated with V. myrtillus plants was dominated by Ascomycota (overall 50% of the total reads), followed by Basidiomycota (overall 15%) and by all the other phyla with percentages below 1% (Glomeromycota, Mortierellomycota, Mucoromycota, Olpidiomycota). A large percentage of the total reads (overall 32%) corresponded to unidentified and not assigned phyla. The phylum Basidiomycota was significantly more abundant in stems than in flowers and leaves ( Supplementary Fig. S2). At the class level (Fig. 2a), Dothideomycetes were the most abundant (overall 29%), followed by Leotiomycetes (14%), Agaricomycetes (11%) and by the other classes with percentages below the 2,1%. Overall, 35% of the total reads corresponded to unidentified and not assigned classes. The classes Leotiomycetes, Dothideomycetes, Tremellomycetes and Agaricomycetes showed significant differences in their abundance across the different organs, as shown in Fig. 2b. In particular, the class Leotiomycetes was significantly more abundant in roots than in all the other organs. At the genus level ( Supplementary Fig. S3), 76% of taxa in all organs were unidentified or not assigned, whereas the most abundant identified genus was Athelia (overall 5%) followed by Phialocephala (overall 3.5%) and Cladosporium (overall 1.8%). The abundance of the Athelia genus was significantly higher in stems ( Supplementary Fig. S3). Among the genera including known ErMF, we found Pezoloma (0.7%), only represented by P. ericae (Supplementary Table S1 Organ-wise comparisons of the relative abundance of fungal orders (Fig. 3) showed that the highest number of significantly different taxa were found when roots were compared with all the other organs. In particular, the orders Helotiales and Leucosporidiales were always more abundant in roots than in the other organs, while Dothideales and Capnodiales, both in the class Dothideomycetes, were less abundant in roots. Sebacinales were more abundant in roots than in leaves and stems. Atheliales were more abundant in stems than in the other organs, while Polyporales were more abundant in leaves and Capnodiales were more abundant in flowers.
The LefSe score (Linear discriminant analysis Effect Size 44 ) was used to estimate differences in the relative taxa abundance among organs at the class, order and genus level (Fig. 4). Few taxa were A correlation network analysis based on Pearson's statistics, which determines whether linear relationships exist between two taxa, showed in the roots a significant co-occurrence of the classes Leotiomycetes, Eurotiomycetes and Geoglossomycetes ( Supplementary Fig. S4). The same analysis at the genus level in the roots showed co-occurrence of genera including known ErMF species, namely within Oidiodendron, Meliniomyces and Serendipita ( Supplementary Fig. S4). In addition, such genera including ErMF species showed a significant co-occurrence with Basidiomycetes known to be ectomycorrhizal on tree species, such as Suillus, Russula, and Lactarius, and with the DSE Phialocephala.
We found 214 core OTUs present in the four organs ( Supplementary Fig. S5). Among them, the most abundant genera were Phialocephala, mainly detected in roots, Athelia, mainly detected in stems, and Cladosporium, mainly detected in flowers. Among the core OTUs were also few genera including known and putative ErMF species, such as Pezoloma (with the single species P. ericae), Meliniomyces (with the two ErMF species M. bicolor and M. variabilis), Geomyces, members of the PAC (Phialocephala, Cadophora) as well as some ectomycorrhizal fungi (Supplementary Fig. S5).
The double-clustering analysis of the core OTUs showed that root and stem samples form distinct clusters, suggesting that the abundance and distribution of the core OTUs changes in these organs, while (not surprisingly) is more similar in flowers and leaves (Fig. 5).   Fig   S6-S7). OTUs attributed to the REA (Supplementary Fig. S7) have been detected mostly in roots, but they were also found in other plant organs.

Plant colonization by Helotiales isolates in vitro.
The 15 Helotiales isolates were tested for their ability to colonize V. myrtillus plants in vitro. The three O. maius strains formed typical coils in the root epidermal cells (Fig. 6a-b-c), whereas the two C. luteo-olivacea colonized the root tissues but did not form specific fungal structures (Fig. 6e-f).
The other Oidiodendron species associated with the roots but did not form typical mycorrhizal coils (Fig. 6d).
We checked for the presence of three of the fungal isolates in the above-ground organs of plants grown in axenic conditions. We extracted total DNA from pooled stems and leaves, using the same protocol described for the field-collected plants and tested it by PCR with primers designed on the fungal ITS2 region (Supplementary Fig. S8). PCR amplification of total DNA extracted from plants tenuissimum isolates produced two amplicons that were attributed by Sanger sequencing to Vaccinium sp. and Oidiodendron spp., respectively (Supplementary Table S3).

Discussion
The plant internal tissues represent a unique ecological niche where some distinctive fungal endophytic species may live. The endophytic association plays an important role in the adaptation to the environment of both plants and fungi, together with the other organisms that constitute the holobiont. It has been suggested that plants select their microbiome for traits rather than taxonomy, because it provides many functions that are a part of an 'accessory genome' and that may be distributed across many different taxa 2 .
Here, we have used a culture-independent approach to investigate the endophytic fungal communities associated with different organs of V. myrtillus (Ericaceae) plants collected in an alpine habitat. At a coarse taxonomic level, the fungal population was dominated by Ascomycetes, followed by Basidiomycetes. This is similar to the results of previous studies on the root-associated fungi of Ericaceae 16,17,19,[34][35][36] but in contrast with the report of Trivedi and colleagues 2 that, based on the analysis of metabarcoding datasets from different angiosperms, stated that the endospheric fungal community was dominated by Basidiomycetes.
Association of some fungal endophytes with specific host tissues has been observed in some plant species 45 . Similarly in V. myrtillus, we showed that the different organs shape the endophytic fungal community. The analysis of beta-diversity revealed that the fungal community colonizing the root endosphere was particularly different from the others, possibly because of the closeness and influence of the rhizopheric soil.
Alpha-diversity indices suggest a similar degree of fungal diversity within the V. myrtillus organs, except for the diversity associated with leaves, that was higher when evenness was taken into consideration by the Shannon index, in line with previous reports 2, 39 .
Relative abundance of lower rank taxa revealed that the Helotiales were more abundant in roots than in the other organs, and Phialocephala and Meliniomyces genera could be considered as biomarkers of the root compartment. In particular, one of the OTUs that determined the divergence of the root compartments from the other plant compartments was assigned to Phialocephala fortinii. This species belongs to the group of the DSE fungi and forms with A. applanata the socalled P. fortinii s.l. -A. applanata species complex (PAC), often found to be associated with Ericaceae roots [15][16][17][18][19] . Meliniomyces comprises species known as ErMF, such as M. bicolor and M. variabilis 28 , both found in our dataset. Sebacinales were also more abundant in roots than in leaves and stems, with Serendipita as biomarker of the root compartment. Sebacinales have been already reported as common fungi in Vaccinium spp. roots 15,19,34 and encompass ubiquitously distributed taxa found as symbionts in diverse mycorrhizal types, ranging from ectomycorrhiza to ericoid and orchid mycorrhiza, and as root endophytes. Species belonging to the Leucosporidiales, found to be more abundant in roots that in the other organs, have been already observed as leaves and stems endophytes both in grasses and in woody plants 46,47 but, to our knowledge, they have never been reported from roots.
Among the dominant genera in the roots, we detected fungal endophytes belonging to the Neonectria genus, as already reported by Zhang and coworkers 18 in blueberry roots. We also identified, in the root compartment, two typically ectomycorrhizal fungal genera, Russula and The plant core microbiota consists of those members of the microbial community that are ubiquitous in the plant compartments. Few dominating taxa in a single V. myrtillus organ turned out to be present, although with lower reads numbers, in the other organs as well, being part of the host core microbiota. This was the case, for example, for the Phialocephala, Athelia and Cladosporium genera. The identification of Phialocephala, represented by the single assigned species P. fortinii, in the core microbiota was interesting, as this DSE fungus is generally reported as a root-specific endophyte 52 .
Members of the core microbiota that can influence the community structure through strong biotic interactions with the host or with other microbial species are defined as 'hub microorganisms' 53 .
Leotiomycetes have been shown to co-occur with Eurotiomycetes in all the plant organs, and in the root with Geoglossomycetes. Leotiomycetes and Geoglossomycetes have been reported as hub taxa in the V. angustifolium root-associated microbiota 35 . These authors suggested an important role of these microorganisms in the wild blueberry soil ecosystem, in particular Leotiomycetes, since this class contains many plant pathogens and mycorrhizal fungi 28 and therefore might influence plant health and the microbiota associated.
Interestingly, the core fungal community of V. myrtillus also included well-established ErMF taxa, such as Pezoloma ericae and Meliniomyces spp., as well as putative ErMF species (Geomyces 54 ). In the core microbiota were also DSE fungal members of the PAC different from Phialocephala, such Although they can promote plant growth in harsh environments 20 , the mode of action of root DSE fungi is elusive 57 , whereas promotion of host growth and fitness by ErMF has been ascribed to plant-fungus interactions occurring at the symbiotic interface formed around the intracellular fungal coils. If these fungi play any role promoting plant survival in the aerial plant compartments, other so far unknown mechanisms may take place.
In conclusion, we have described by metabarcoding the diversity of fungi associated with the endosphere of below-ground and, for the first time, above-ground organs of V. myrtillus. The results have significantly increased our knowledge of the V. myrtillus fungal microbiota and revealed that fungal strains so far considered as strict root symbionts can occupy different niches within the plant, as they were detected in above-ground organs as well.
Several examples of fungi displaying dual life niches have been reported 58 . In particular, ErMF were already known to behave as dual saprotrophs/symbionts, with different root-interacting strategies according to the plant hosts 41 . Here, we show that they may occupy a further ecological niche as stem/leaves endophytes. This hypothesis will require further investigations, such as the isolation of ErMF from field-collected V. myrtillus stems/leaves and/or plant inoculation in vitro 59 .

Sampling site and description
The sampling site (45°50'40'' N, 7°34'41'' E, 2200 m a.s.l.) was a subalpine meadow, unused for 10 years, associated with the ICOS network (Integrated Carbon Observation System; station ID: IT-Tor) and managed by ARPA Valle d'Aosta (Regional Agency for the Environment Protection). In

Bioinformatics
Sequencing adapters and primers were removed and then paired-end reads from each sample were merged with Pear v.0.9.2 60 using a quality score threshold set at 28 and a minimum length after trimming set at 200 bp. The assembled reads were then processed using the Quantitative Insights into Microbial Ecology (Qiime) v.1.9.1 software package 61 . Sequence processing and sample assignment were performed with a minimum sequence length cut-off of 200 bp and a Phred quality score of 28, calculated over a sliding window of 50 bp. Chimeric sequences were removed performing a de novo detection using UCHIME 62 .OTUs were obtained using VSEARCH 63

Isolation of endophytes from roots
Roots were homogenized in sterile water by a sterile glass potter. The homogenized root suspension was centrifuged and washed three times with distilled sterile water; the supernatant was then discarded, and the pellet was suspended in sterile water and plated on MEA medium (2% malt extract, 1,8% agar) amended with antibiotics (15 mg/l streptomycin and 50 mg/l chloramphenicol).
The plates were incubated at 25°C and as soon as fungal colonies appeared, they were individually transferred to fresh plates for subsequent identification by morphological and/or molecular analyses. Molecular identification was performed by genomic DNA extraction, followed by PCR amplification of the ITS2 region, Sanger sequencing and Blast search on both NCBI nucleotide library and UNITE database. Some of the isolated strains have been deposited at the Mycotheca Universitatis Taurinensis -MUT-collection of the University of Turin, Italy; see Table S2). Blast search of the ITS2 sequences of a few fungal isolates placed them in the Helotiales. A more precise assignment of these isolates in the Helotiales was supported by the phylogenetic analysis performed according to published methods 29

Colonization of V. myrtillus plants in vitro
The Helotiales strains isolated in this work and an O. maius isolate already characterized for its ErM forming abilities (OmMUT1381, deposited at the MUT) were co-cultivated with V. myrtillus seedlings according to a standardized protocol for mycorrhizal synthesis 69 . Root colonization by fungal hyphae of six isolates, three of them expected to be mycorrhizal, was monitored by bright field microscopy after staining with 0.1 % (w/v) cotton blue and destaining overnight with 80% lactic acid.
Fungal colonization of the above-ground organs was checked by PCR, following the same protocols used for DNA extraction and PCR amplification of the fungal ITS2 region from field collected plants (see above). Genomic DNA from OmMUT1381, OmMUT1348 70 , a basidiomycete and V. myrtillus were used as controls in the PCR reaction.

Data availability statement
The raw sequences from the metabarcoding experiment have been deposited with the BioProject ID PRJNA769432 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA769432). The data-sets generated and analyzed during the current study are included in this published article (and its Supplementary Information files).

Competing interests
The authors declare no competing interests.      Beta-diversity of the fungal communities associated with the different plant organs. The beta-diversity among the different organs was estimated by a NMDS analysis based on Bray-Curtis dissimilarities, with the following parameters: taxonomic level: feature, statistical method: PERMANOVA, experimental factor: organ. Fi=Flowers, Fo=Leaves, Fu=Stems, R=Roots. C1-C5: samples.   Heat tree matrix depicting the different taxa abundance among the plant organs, for all orders in the dataset. The size of the nodes in the gray cladogram (right) represents the number of OTUs identi ed at that taxonomic level. The small cladograms show the pairwise comparisons among the organs: a yellow node indicates a higher abundance of the taxon in the organ indicated in yellow, than in the organ indicated in green. A green node indicates the opposite. Taxa identi ed as differently represented, statistically supported by the Wilcoxon test (p<0.05), are tagged with a white asterisk.  Heatmap of the core OTUs genera. A double clustering based on average linkage algorithm and Pearson correlation, that clusters together features or samples with similar behavior, has been performed.