Breaking boundaries: fungi in the “rhizoctonia” species complex exhibit systemic colonization in three terrestrial orchid species

Tulasnella, Ceratobasidium , Serendipita , fungal endophytes, orchid mycorrhiza 37 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 3

38 39 All plants share their lives with a complex and diverse microbial community that contributes to plant growth 40 and defense against biotic and abiotic stress (Hardoim et al., 2015; Almario et al., 2017). Fungi are important 41 components of the plant microbiota and can colonize the external surfaces as well as the internal tissues of all 42 plant organs, both below -ground and above -ground (Choi et al ., 2021). The root -associated mycobiota 43 comprises mycorrhizal fungi, a diverse group of symbiotic fungi that form specialized plant-fungus interfaces 44 inside the roots of most land plants (Smith & Read, 2008). Mycorrhizal symbioses are thought to have been 45 instrumental for land colonization by early plants, and their ecological success and wide distribution are likely 46 related to the improved mineral nutrition and health of the host plant (Genre et al., 2020). Most plants benefit 47 from the mycorrhizal association, some plant species, like orchids, being particularly reliant on these symbiotic 48 fungi for their survival. 49 Orchids belong to one of the largest families of flowering plants, counting more than 28,000 terrestrial 50 and epiphytic species distributed in extremely diverse habitats, ranging from tropical forests to semi -arid 51 deserts (Chase et al., 2015; Christenhusz & Byng, 2016). Despite their diversity, survival of all orchids in 52 nature is intrinsically bound to the association with orchid mycorrhizal (OrM) fungi. All orchids produce tiny 53 “dust” seeds devoid of stored nutrients and with an immature embryo (Arditti & Ghani, 2000). Orchid seeds 54 that successfully germinate develop a protocorm, a postembryonic heterotrophic structure that precedes 55 seedling formation (Arditti & Ghani, 2000; Rasmussen, 1995). OrM fungi are necessary in these early stages 56 because they induce seed germination and provide germinating seeds and protocorms with organic carbon and 57 other essential nutrients (Smith & Read, 2008; De Rose et al., 2023). Mycorrhizal orchid protocorms eventually 58 develop into adult plants by forming aerial organs that can be achlorophyllous or chlorophyllous. 59 Achlorophyllous orchids lack photosynthesis and remain completely dependent on their OrM fungal symbionts 60 for organic carbon supply, a strategy termed full mycoheterotrophy (Hynson et al., 2013). However, stable 61 isotope natural abundance indicates that green orchids can also receive carbon from their symbiotic fungal 62 partners. In particular, green orchids living in shady forest habitats can supplement inefficient photosynthesis 63 with fungal-derived carbon, a strategy termed partial mycoheterotrophy or mixotrophy (Selosse & Roy, 2009; 64 Merckx, 2013). Even photosynthetic orchids living in open meadows, expected to be fully autotrophic, can use 65 OrM fungi to supplement their carbon demands in some cases (Gebauer et al., 2016; Girlanda et al., 2011; 66 Schiebold et al., 2018). Thus, OrM fungi are critically important for seed germination, seedling survival and 67 plant growth in nature, and it is therefore not surprising that local‐scale distribution and population dynamics 68 of orchids can be limited by distribution and abundance of their symbiotic OrM fungi (McCormick & 69 Jacquemyn, 2014; McCormick et al., 2018). 70 OrM fungi belong to diverse taxa, whose phylogenetic position mirrors the habitat and the trophic 71 abilities of the host plants. Terrestrial achlorophyllous and green orchids restricted to forest floors usually form 72 specific associations with fungi also capable of forming ectomycorrhiza (ECM) on photosynthetic trees 73 (Dearnaley et al., 2012), but associations with wood-decomposers have been also reported (Ogura-Tsujita et 74 al., 2021). Photoautotrophic orchids mainly associate with OrM fungi belonging to the ‘rhizoctonia’ species 75 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 4 complex, a polyphyletic group comprising teleomorphs in three distinct families of Agaricomycotina 76 (Basidiomycota): Tulasnellaceae and Ceratobasidiaceae in the Cantharellales, and Serendipitaceae in the 77 Sebacinales (Roberts, 1999; Taylor et al., 2002; Weiß et al., 2004, 2016). The biology and ecology of 78 rhizoctonia-like OrM fungi are poorly understood when compared with ECM fungi, also because of their 79 inconspicuous nature (Roberts, 1999). An intriguing feature of rhizoctonia -like OrM fungi is that some 80 members are not uniquely mycorrhizal, as they have been found as non-mycorrhizal endophytes in the roots 81 of non-orchid plants, where they can promote plant growth and tolerance to biotic and abiotic stress (Lahrmann 82 et al., 2015; Sarkar et al., 2019; Mahdi et al., 2022; Mosquera -Espinosa et al., 2013; Ray et al., 2018). For 83 example, the OrM fungus Serendipita (= Sebacina or Piriformospora) vermifera, originally isolated from 84 mycorrhizal roots of terrestrial Australian orchids in the genus Caladenia (Warcup and & Talbot, 1967), is 85 considered a generalist root endophyte because it can colonize a wide range of monocots and dicots without 86 forming typical mycorrhizal fungal coils (Weiß et al., 2016; Qiang et al., 2012 ; Unnikumar et al., 2013). A 87 related species, Serendipita indica, improves the availability of nitrogen (Saleem et al., 2022) and produces 88 phosphatases and organic acids which contribute to solubilization of phosphate from insoluble polyphosphates 89 and organic phosphates (Johnson et al., 2014). In soybean, S. indica improved N, P and K uptake as a result 90 of upregulation of transporter genes related to P and N uptake (Bajaj et al., 2018). It also produces plant growth 91 regulators that affect the root architecture, such as indole acetic acid (IAA, the most common natural auxin) 92 and cytokinins, which have a positive effect on root growth (Oelmüller et al., 2009; Liu et al., 2023). Other 93 OrM rhizoctonia-like fungi have been found in the roots of non-orchid plants, although their potential role in 94 non-orchid hosts is unknown (Selosse et al., 2022). For example, the same Tulasnella sp. OTU (Operational 95 Taxonomic Unit) was identified in the roots of Orchis purpurea and of a nearby Bromus erectus plant in a 96 Mediterranean meadow (Girlanda et al., 2011). 97 Saprotrophic capabilities have been often reported for OrM rhizoctonia -like fungi grown in vitro, 98 where these fungi can produce a range of plant cell wall degrading enzymes (Nurfadilah et al., 2013, Zhao et 99 al., 2021; Novotná et al., 2023). In agreement with these observations, the sequenced genome of two OrM 100 fungi, a T. calospora and a S. vermifera isolate, has revealed an expanded family of Carbohydrate Active 101 Enzymes (CAZymes) mainly involved in cellulose degradation (Kohler et al., 2015). This rich repertoire of 102 plant cell wall degrading enzymes would suggest that rhizoctonia-like OrM fungi can spend part of their life 103 cycle growing saprotrophically on plant litter in soil. However, their actual saprotrophic capabilities in nature 104 are unclear and a limited saprotrophic potential is suggested, at least for some OrM fungi, by the results of 105 metabarcoding investigations on the occurrence of rhizoctonia-like OrM fungi in the soil surrounding the roots 106 of their orchid host. In particular, OTUs identified in the mycorrhizal roots of terrestrial orchids in Italy 107 (Voyron et al., 2017), in Australia (Egidi et al., 2017), in the United States (Kaur et al., 2019) and in Denmark 108 (Hartvig et al., 2024) could not be detected in the soil outside the orchid rhizosphere. As for S. vermifera, an 109 experiment set up to evaluate fungal transmission to plants that can harbor this fungus as beneficial root 110 endophyte (Ray et al., 2018) showed that transmission failed when the roots of different individuals were kept 111 separate, thus indicating that migration of this fungus in root-free bulk soil is unlikely. Thus, the poor success 112 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 5 of some OrM fungi in the “rhizoctonia” species complex as soil colonizers, as well as their potential behavior 113 as root endophytes, raise intriguing questions on the actual niche of these fungi. Moreover, a recent study 114 aimed at investigating the potential environmental reservoir of OrM fungi for S. spiralis (Calevo et al., 2021) 115 showed that, despite the removal of environmental compartments (neighbouring plants and soil), a core of 116 OrM fungi was able to colonize newly emergent roots, suggesting an internal transmission starting from either 117 older roots or other orchid tissues. This finding further supports the hypothesis that some OrM fungi could be 118 strictly associated to orchid tissues, spending most of their life cycle inside the plant and therefore qualifying 119 as “ecologically obligate” orchid symbionts (Calevo et al., 2021) . 120 Mycorrhizal fungi are reported to be strictly associated with the roots of land plants (Genre et al., 121 2020). However, given the peculiarities of OrM fungi in the “rhizoctonia” species complex, we have tested the 122 hypothesis that at least some of these OrM fungi may use the whole plant as an ecological niche by colonizing 123 not only the roots, but also other plant organs. Interestingly, rhizoctonia -like fungi were isolated from both 124 roots and leaves of orchid species in the genus Lepanthes (Bayman et al., 1997) and in Acampe praemorsa, 125 Cymbidium aloifolium and Vanda testacea (Behera et al., 2013). However, the phylogenetic position of these 126 fungal isolates was not assessed, and it is therefore unclear whether roots and leaves hosted the same fungi. 127 Here, we have used a metabarcoding approach to detect the occurrence of rhizoctonia -like taxa related to 128 known OrM fungi in the mycobiota amplified from vegetative organs (roots, stems and leaves) of three 129 terrestrial orchid species: Spiranthes spiralis, Serapias vomeracea and Neottia ovata. In one of the three orchid 130 species, S. spiralis, we also investigated the seed mycobiota to test the possibility of a vertical transmission of 131 OrM fungi. 132 133

134 135 Orchid species 136 137 The plant species investigated in this work were Spiranthes spiralis, Serapias vomeracea and Neottia ovata 138 (Fig. 1). 139 140 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 6 141 Figure 1 . Floral morphology of the three target orchid species: (A) Spiranthes spiralis , (B) Serapias 142 vomeracea, and (C) Neottia ovata . Each panel illustrates the distinct floral characteristics unique to each 143 species, highlighting their morphological diversity. 144 145 Spiranthes spiralis (Subfamily Orchidoideae, Tribe Cranichideae) is a herbaceous orchid that flowers 146 in late summer -autumn with a particular spiral -shaped inflorescence. It is widely distributed in Southern 147 Europe and in the Mediterranean region, where it grows in pine, oak, chestnut, hornbeam and birch forests, 148 dry meadows as well as in flat grasslands and semi-rocky areas. The preferred substrate is both calcareous and 149 siliceous, with neutral pH. New leaves, formed at the same time or after the flower stem, stand together in a 150 rosette beside the stem; the rhizome is periodically generated every year with new roots and stems (Arditti, 151 2002). 152 Serapias vomeracea (Subfamily Orchidoideae, Tribe Orchideae) is a bulbous herbaceous plant, with 153 two underground globose rhizotubers and erect stems of purplish-vinous color, varying in height from 20 to 154 60 cm. The inflorescence, loose and elongated, is composed of a few spaced flowers. It can be found in sunny 155 and wet meadows, on the edges of paths, in bushy environments from the plain up to 1200 m of altitude. It is 156 the most widespread species in the genus Serapias and is distributed in most Europe (Arditti, 2002). 157 Neottia ovata (Subfamily Epidendroideae) is a perennial rhizomatous orchid regularly found in a wide 158 range of habitats including woods, shrubs, hedges, calcareous pastures, dunes and marshes and, to a lesser 159 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 7 extent, meadows. It grows on acid and calcareous substrates in damp and cold woods, mainly conifers, on 160 sphagnum moss and carpets, often together with blueberry ( Vaccinium myrtillus) from 900 to 2100 m a.s.l. 161 The lower capsules in the inflorescence can mature and disperse the seeds even before the flowers placed 162 higher in the inflorescence are pollinated. It is common throughout Europe (Arditti, 2002). 163 164 Collection and Sterilization of Plant Samples 165 166 Various parts of the three orchid species were collected in the fall of 2016 and spring of 2017 from different 167 locations within the provinces of Imperia ( Serapias vomeracea), Savona (Spiranthes spiralis), and Genova 168 (Neottia ovata) in Liguria, Italy. These parts included roots, stems, leaves, and seed pods. Spiranthes spiralis 169 was collected twice: samples S. spiralis_1 were collected in 2016 (Calevo et al., 2021) and S. spiralis_2 were 170 collected in 2017 to include capsules and seeds. Once collected, plant samples were immediately kept on ice 171 within sterile containers and transported to the lab for storage at 4°C prior to examination. Sterilization of 172 orchid tissues was performed as described in Alibrandi et al. (2021). Briefly, the above -ground parts of the 173 plants underwent a sequential sterilization process involving a one-minute soak in 70% ethanol, a two-minute 174 treatment with 2.5% sodium hypochlorite, and another one -minute immersion in 70% ethanol. This was 175 followed by five washes in sterile water. The roots were first cleaned with sterile water, subjected to sonication, 176 and then sterilized using 95% ethanol for 20 seconds and 5% sodium hypochlorite for three minutes, with 177 seven subsequent rinses in sterile water. To verify the effectiveness of the sterilization, the final rinse water 178 and imprints from the sterilized plant surfaces were cultured on various agar media (Luria Bertani - LB, King’s 179 B) and monitored for microbial growth over a period of 4 -7 days at 28°C. 180 181 DNA Isolation, amplification and sequencing 182 183 Total DNA was isolated from approximately 100 mg of tissue, using the DNeasy Plant Mini Kit from 184 QIAGEN, following the guidelines provided. DNA integrity and concentration were determined using a 185 spectrophotometer (ND-1000 Spectrophotometer NanoDropH; Thermo Scientific, Wilmington, Germany). 186 The ITS2 region of the nuclear ribosomal DNA was amplified from the extracted DNA through a semi-nested 187 PCR method. Initially, the full ITS region (ITS1 -5.8S-ITS2) was amplified using primer pairs specifically 188 designed for orchid mycorrhizal fungi (Taylor & McCormick, 2008), ITS1 -OFa, ITS1-OFb and ITS4 -OF 189 primers, and primers ITS1 and ITS4tul (Tul). A nested PCR was then conducted to amplify the ITS2 region 190 using tagged primers ITS3mod and ITS4 (White et al., 1990). All amplifications were performed in three 191 replicates. The PCR reactions were carried out with mixture and thermal cycling conditions as described in 192 Calevo et al. (2020). The resulting PCR products were verified on a 1% agarose gel, replicates pooled together 193 and purified with a Wizard SV Gel and PCR Clean -Up System (Promega) , following the manufacturer’s 194 instructions. Quantification was performed with QUBIT 2.0 (Thermo Fisher Scientific, Waltham, MA, USA) 195 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 8 before preparing sequencing libraries, which were then sequenced using Illumina MiSeq technology (2 x 250 196 bp) by IGA Technology Services Srl (Udine, Italy) . 197 198 Sequence analyses 199 200 The initial step in analyzing the sequencing data involved merging the paired -end reads from each sample 201 collection using PEAR v.0.9.2 (Z hang et al., 2014), setting a quality score cutoff at 28 for trimming and 202 establishing a minimum read length of 200 base pairs post-trimming. The merged reads were then processed 203 with the Quantitative Insights into Microbial Ecology (QIIME) v.1.8 software suite (Caporaso et al., 2010), 204 adhering to specific criteria for sequence length, quality score, and primer mismatch tolerance as described in 205 Voyron et al. (2017). Chimeric sequences were identified and excluded using an abundance-based approach 206 with USEARCH61 (Edgar, 2010) within the QIIME framework. Clustering into operational taxonomic units 207 (OTUs) was performed using a reference -guided method with a 98% similarity threshold, retaining only 208 clusters with a minimum of 10 sequences. The UNITE database served as the reference for OTU identification 209 and taxonomic classification (Abarenkov et al., 2010; Koljalg et al., 2013; http://unite.ut.ee, last accessed 25 210 May, 2019), employing the BLAST algorithm (Altschul et al., 1990) with a set e-value threshold of 1e-5. The 211 most prevalent sequences within each OTU (the OTU representative sequences) of putative OrM fungi and 212 fungi occurring in ≥80% plants in at least one orchid species were submitted to GenBank and recorded under 213 the following string of accession numbers: PQ644909 - PQ645014 . 214 We carried out the maximum likelihood (ML) analyses with representative sequences from 215 ceratobasidioid, tulasnelloid and sebacinoid s.l. OTUs. Sequences that were the closest matches from BLAST 216 searches, as well as sequences from a diverse range of terrestrial orchids, including the species under study, 217 from various global locations and habitats, and sequences from other plants and fungal specimens were 218 included in the analyses. The sequences were aligned using MAFFT (Katoh & Toh, 2010) with default settings, 219 and the alignments were manually edited in MEGA v.7.0.26 (Kumar et al., 2016). ML estimations were 220 conducted using RAxML v.8 (Stamatakis, 2014) over 1000 bootstrap replicates (Felsenstein, 1985), utilizing 221 the GTR + GAMMA algorithm. The best tree topology was determined on the CIPRES Science Gateway 222 (Miller et al., 2011); support values from bootstrapping runs were mapped on the globally best tree using the 223 –f option of RAxML and -x 12345 as a random seed. Nodes with bootstrap support below 70% were not 224 considered robust. 225 226 Statistical analyses 227 228 Comparative Analysis of Root Sample Datasets 229 230 To facilitate comparative analysis across datasets derived from various tissue samples, including the newly 231 collected Spiranthes spiralis and previously gathered tissues at the initial time point, a standardization process 232 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 9 was implemented. This involved normalizing the sequencing depth to a consistent number of sequences (1000) 233 for each sample, utilizing the rarefy_even_depth function within the ‘phyloseq’ package in R (R Core Team, 234 2021). 235 Analysis of Fungal Community Composition 236 237 The distribution of OTUs across different tissue categories was statistically compared using chi-squared tests 238 to determine if there were significant differences in the proportions of OTUs recovered. 239 The impact of orchid tissue on the composition of OrM fungal communities was investigated through 240 permutational multivariate analysis of variance (PERMANOVA), employing 999 permutations within the 241 adonis function of the ‘vegan’ package in R (Oksanen et al., 2013; R Core Team, 2021). Prior to this, the 242 homogeneity of multivariate dispersions among groups was evaluated using the betadisper and permutest 243 functions, also within the ‘vegan’ package (Oksanen et al ., 2013). Constrained Analysis of Principal 244 Coordinates (CAP) was also performed to evaluate the effects of species identity, organ type, and their 245 interaction on OrM fungal community composition. CAP was chosen due to its robustness to heterogeneity in 246 multivariate dispersions, as indicated by beta dispersion tests, which revealed significant differences in 247 dispersion for organ type ( P = 0.0382) but not for species ( P = 0.1503). 248 Additionally, to visualize the distribution and relationships of OrM fungi, we employed R (R Core 249 Team, 2021) packages such as ‘ggplot2’ (Wickham, 2016), ‘ggdendro’ (de Vries & Ripley, 2023), and 250 ‘reshape2’ (Wickham, 2020) to create a heatmap of OrM fungi frequency in orchid tissues. This heatmap 251 displayed hierarchical clustering of orchid samples based on the frequency patterns of OrM fungi association. 252 To visualize OrM OTUs shared among different orchid tissues we plotted OTUs presence and frequency in 253 chord diagrams generated using the ‘circlize’ package (Gu et al., 2014) for visualizing relationships, with 254 layout adjustments made using the ‘gridExtra’ package (Auguie, 2017), and Venn diagrams. 255 256

257 258 After filtering and cleaning the Illumina MiSeq reads, we obtained 8,178,658 high-quality sequences that were 259 clustered in 1353 OTUs (98% sequence identity). Only OTUs identified in the kingdom Fungi when blasted 260 on the UNITE database and represented by at least 10 reads were considered for further analyses, for a total of 261 2,379,265 reads in 761 OTUs. 262 263 The mycobiota in the vegetative organs of the three Mediterranean orchid species 264 265 The fungal OTUs retrieved from the vegetative organs of the three orchid species were mostly assigned 266 to Ascomycota (471 OTUs, 51.4% of total OTUs), followed by Basidiomycota (297 OTUs, 32.4% of total 267 OTUs) and unidentified OTUs (116 OTUs, 12.7% of total OTUs). Only very few OTUs were assigned to 268 Mortierellomycota, Glomeromycota, Mucoromycota, Chytridiomycota, Kickxellomycota, Olpidiomycota and 269 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 10 Rozellomycota, according to NCBI and UNITE taxonomic classification (each of the latter phyla accounting 270 for <1.0% of total OTUs). 271 The mycobiota differed both among plant parts and among orchid species. In S. spiralis _1, S. 272 vomeracea and N. ovata, the number of OTUs shared by at least two vegetative organs was significantly lower 273 than the number of OTUs unique of a single organ (P < 0.00001, chi -squared tests). In all orchid species, in 274 particular, roots and leaves in each plant shared a greater proportion of taxa with stems than with each other 275 (Fig. S1). Generally, higher OTU richness was found in leaves than in roots (except in S. spiralis_2, in which 276 OTU numbers did not differ significantly between leaves and roots; P < 0.00001, chi-squared tests). Despite 277 the differences in OTU profiles between roots and leaves, most OTUs (394, 51.2% of total OTUs) were 278 retrieved in both organs of the same individual orchid plant (70.1% of them being found in all vegetative 279 organs of the same plant). Other OTUs (124 OTUs, 16.1%) were retrieved in either roots or leaves of different 280 individuals of the same orchid species . 281 The different orchid species featured a different mycobiota. In fact, irrespective of the vegetative 282 organ, a higher number of OTUs was exclusively found in a single orchid species (65.8-71.4% of total OTUs) 283 than in more than one orchid species (P < 0.00001, chi -squared tests). In the PCA space, OTU profiles 284 segregated based on orchid species rather than organ type (Fig. S2), with a partial overlap between the OTU 285 profiles of S. vomeracea and N. ovata . A more distinct profile in both samplings characterized S. spiralis, 286 especially when presence/absence was considered (Jaccard index) . 287 Seventy-one OTUs (49.3% Ascomycota, 47.9% Basidiomycota, and one Mucoromycota) occurred in 288 ≥80% samples of at least one organ in at least one orchid species (Table S1). Basidiomycota OTUs mostly 289 included Cantharellales (35.3% of Basidiomycota OTUs, comprising several “rhizoctonias”), followed by 290 Polyporales (15.2%) such as Bjerkandera adusta, Daedaleopsis confragosa, Lentinus tuber-regium, Trametes 291 versicolor . 292 Clustering heatmap based on the dominant non -rhizoctonia OTUs revealed variable occurrence 293 patterns of these OTUs in the different orchids (Fig. S3). Samples clustered again according to orchid species, 294 OTU profiles in roots and stems being closely related in all orchids. Some taxa were retrieved in all vegetative 295 organs of all orchids and included Cladosporiaceae (Ascomycota, Dothideomycetes, Capnodiales; OTU 127 296 Mycosphaerella tassiana , OTU 1180 Cladosporium arthropodii) and other Dothideomycetes (OTU 1105 297 Aureobasidium pullulans, OTU 11092 Didymella exigua, OTU 11092 Pyrenochaetopsis leptospora), as well 298 as Bjerkandera adusta (OTU 733; Basidiomycota, Agaricomycetes, Polyporales, Meruliaceae). Other OTUs 299 occurred in all organs of at least one orchid, while lacking in other species (e.g. OTUs 460 Pholiotina brunnea 300 and 290 Sistotrema brinkmannii in S. spiralis_1, OTU 209 Bifiguratus adelaidae in S. vomeracea and N. ovata) 301 (Fig.S3, Table S1). 302 303 Potential OrM fungal taxa in S. spiralis, S. vomeracea and N. ovata 304 305 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 11 Previous investigations on S. spiralis (Tondello et al., 2012; Duffy et al., 2019; Calevo et al., 2021), S. 306 vomeracea (Girlanda et al., 2011) and N. ovata (Jacquemyn et al., 2015; Těšitelová et al., 2015; Wang et al., 307 2023) revealed that rhizoctonia -like fungi that include tulasnelloid, ceratobasidioid and sebacinoid s.l. 308 (sebacinoid s.s. and serendipitoid) fungi are the dominant OrM symbionts in these three terrestrial orchid 309 species. We searched therefore, in the general database (Table S1), OTUs identified by Blast searches in the 310 Cantharellales and Sebacinales, and selected 46 OTUs of potential OrM fungi belonging to tulasnelloid (11 311 OTUs), ceratobasidioid (27 OTUs) and sebacinoid s.l. fungi (9 OTUs). Representative sequences of these 312 different OTUs were blasted against the NCBI nucleotide database. The same sequences were also aligned 313 with reference sequences to better define their phylogenetic affinities, and the corresponding Maximum 314 likelihood trees are shown in Figs. S4, S5 and S6 for tulasnelloid, ceratobasidioid and sebacinoid s.l. fungal 315 OTUs, respectively. 316 With a single exception (OTU 184), the tulasnelloid OTUs clustered in a single clade (supported by 317 99% bootstrap) along with sequences previously obtained from the roots of S. spiralis (Calevo et al., 2021) 318 and from other orchid species (i.e. from Orchis sspp., Cachapa Bailarote et al., 2012; Pecoraro et al., 2018; 319 Calevo et al., 2020). Sequences in this tulasnelloid cluster were included in ‘species hypotheses’ of the UNITE 320 database (Kõljalg et al., 2013) (SH1144039.08FU and SH1507552.08FU -DOI: SH1507552.08FU, Kõljalg et 321 al., 2021) and assigned to Tulasnella helicospora. OTU 184 clustered separately, together with other sequences 322 from S. spiralis (Duffy et al., 2019; Calevo et al., 2021) or other orchids (i.e., Liebel et al., 2010; Girlanda et 323 al., 2011; Waud et al., 2014; Jacquemyn et al., 2015) as well as sequences from the soil of one of the study 324 areas (Voyron et al., 2017; Fig. S4. Similarly, ceratobasidioid and sebacinoid s.l. OTUs were phylogenetically 325 closely related to fungi identified in either the same or different orchid species, in roots of non-orchid plants 326 or in the soil of one of the study areas (Figs. S5 and S6). Based on the clustering patterns in these phylogenetic 327 analyses, the original 11 tulasnelloid, 26 ceratobasidioid and 9 sebacinoid s.l. OTUs were reduced respectively 328 to 2, 23 and 8 OTUs for subsequent statistical analyses (see below). 329 330 Many putative OrM fungi colonize the three orchid species systemically 331 332 Each plant hosted 10.9 ± 4.4 putative OrM fungal OTUs (mean ± standard deviation; 5-19 depending on the 333 orchid individual). In each orchid species, ≥80% of plants hosted several ceratobasidioid and/or tulasnelloid 334 fungi (Fig. 2, Table S1). All OTUs occurred in orchid roots (77.4% of them being retrieved in ≥60% plants in 335 at least one orchid species), with the exception of the sebacinoid s.l. OTUs 1014 and 1154, which were only 336 retrieved in stems of S. vomeracea and S. spiralis _2, respectively. 337 338 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 12 339 Figure 2. Heatmap and hierarchical clustering of OrM OTU frequency distribution across plant species and 340 organs. The heatmap displays the frequency percentage (0 -100%) of different OTUs in stem, leaf, and root 341 samples from the target orchid species ( Spiranthes spiralis_1, Spiranthes spiralis_2, Serapias vomeracea, 342 Neottia ovata). The dendrogram on the right shows the hierarchical clustering of samples based on their OTU 343 composition similarity. Color intensity from white to dark blue represents increasing OTU frequency . 344 345 346 To test our hypothesis that at least some OrM fungi may colonize their orchid host systemically, we 347 traced the presence and distribution of the 33 OTUs assigned to sebacinoid s.l., tulasnelloid and ceratobasidioid 348 fungi in the vegetative organs of the three orchid species. The results are summarized in the Venn diagrams in 349 Fig. S7, that also reports the list of shared OTUs. A percentage of OTUs ranging from 16.7% in S. vomeracea 350 to 50.0% in N. ovata was found in roots, leaves and stems, indicating that these fungi are able to colonize all 351 the vegetative plant organs. In particular, a broad majority of the OrM fungi found in roots (84.8 %) was also 352 retrieved from either stems or leaves in the same orchid plant, 61.5 % being found in all vegetative organs of 353 the same plant. Although OrM fungi occurred more often in roots than in either stems or leaves (P = 0. 013131 354 and P < 0.00001, respectively), and more frequently in stems than in leaves (P = 0.004819, chi-squared tests; 355 Fig. S8), they were detected more often in more than one organ than in a single one (P < 0.00001, chi-squared 356 tests). Even more so, they were found more frequently in all three organs of the same plant than in any 357 combination of two organs (P = 0.000027, P < 0.00001 and P < 0.00001 for the combinations roots&stems, 358 roots&leaves and stems&leaves, respectively, chi-squared tests), or any single organ (P < 0.00001 for either 359 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 13 roots, stems or leaves, chi-squared tests). Only the sebacinoid s.l. OTU 1070 was exclusively found in roots, 360 but it was extremely sporadic, being retrieved from a single S. vomeracea plant. 361 The chord diagram in Fig. 3 shows , in the three orchid species, the distribution of tulasnelloid, 362 ceratobasidioid and sebacinoid s.l. fungi in all organs of the same plant; for instance, the tulasnelloid OTU 162 363 assigned to T. helicospora , the ceratobasidioid OTUs 222, 254, 274_867, 263_319_1569, 275 and 907, and 364 the sebacinoid s.l. OTU 458 were found in all organs of all individuals in at least one orchid species (Fig. 3 a-365 c). On the other hand, OTU distribution profiles could differ among organs (e.g. between leaves and either 366 roots or stems in S. spiralis _2; P = 0. 00572 and P = 0. 0236, respectively, Kruskal -Wallis tests). 367 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 14 368 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 15 369 Figure 3. Circular diagrams illustrating the distribution of rhizoctonia-like OTUs (Operational Taxonomic 370 Units) across different organs of the same plant for the three species: (A) Spiranthes spiralis_2, (B) Serapias 371 vomeracea, (C) Neottia ovata. Each diagram shows connections between OTUs and plant organs (root, leaf, 372 stem, capsule, seed), with color-coded segments representing different organs. The width of the connections 373 indicates the abundance of OTUs in each organ, highlighting the diversity and distribution patterns within each 374 species. Blue arches indicate ceratobasidioid OTUs, green arches tulasnelloid OTUs, while yellow arches 375 indicate sebacinoid s.l. OTUs. 376 377 Like the general mycobiota, the OrM fungal assemblages in the different samples were significantly 378 shaped by both the plant species and the plant organ (PERMANOVA, P < 0.001, Table S2). However, when 379 subjected to NMDS analysis (3Dstress = 0.1298; linear fit, R2=0.81; Fig. 4), orchid samples mostly grouped 380 according to orchid species rather than plant organ. In fact, different orchids featured different OrM fungi 381 distribution profiles. For instance, N. ovata featured a lower proportion of occurrences only in roots, and a 382 higher proportion of occurrences in leaves, than S. spiralis_1 (P = 0.048819 and P < 0.00001, respectively, 383 chi-squared tests). S. spiralis_2 featured a lower number of OrM fungal OTUs per plant than S. spiralis_1 (P 384 = 0.01762, Kruskall-Wallis test). OTUs which were exclusively found in a single orchid accounted for 41.9-385 58.1% of total OTUs in either roots, stems or leaves. The OrM fungal assemblage of S. spiralis in the first 386 sampling (S. spiralis_1) appeared quite different from the profile of the second sampling (S. spiralis_2), mainly 387 because of the higher frequency of tulasnelloid OTUs in the first sampling and the different distribution profiles 388 of ceratobasidioid OTUs (see e.g. OTUs 241, 274 and 907, Fig. 2). Based on OTU frequencies, stem samples 389 clustered with either root or leaf samples of the same orchid, while root and leaf samples never clustered 390 directly together (Fig. 2). 391 392 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 16 393 Figure 4. Non-metric multidimensional scaling (NMDS) plot of plant organ samples based on species. This 394 NMDS plot visualizes the relationships among different plant organs (leaf, root, stem) based on OrM OTUs 395 similarities across four species: Neottia ovata, Serapias vomeracea, Spiranthes spiralis _1, and Spiranthes 396 spiralis_2. The axes represent NMDS1 and NMDS2 dimensions, with a stress value of 0.129818 indicating a 397 good fit for the ordination. Data points are shaped according to organ type and colored by species. 398 399 Colonization of reproductive organs by putative OrM fungi 400 401 The OrM fungal occurrence and distribution profiles were also assessed in the reproductive structures (capsules 402 and/or seeds) of a subset of plants. Twenty-three tulasnelloid, ceratobasidioid and sebacinoid s.l. OTUs were 403 retrieved from capsules/seeds of these plants, none of them being exclusively found in these reproductive 404 structures (Fig. 3a-c). In fact, in each of the three orchid species, the OTUs retrieved from both vegetative 405 organs and reproductive structures were found more often in more than one organ from the same plant than in 406 capsules only (P = 0.001094, < 0.00001 and < 0.00001 for S. vomeracea, N. ovata or S. spiralis_2, respectively, 407 chi-squared tests), or just in seeds in S. spiralis_2 (P < 0.00001, chi-squared tests). In the latter samples, OrM 408 fungi occurred more frequently in both capsules and seeds than in either structure type alone (P < 0.00001 and 409 0.000639 for capsules and seeds, respectively, chi -squared tests). Only four OTUs (the two ceratobasidioid 410 OTUs 340 , Fig. 3b, and 825, Fig. 3c, and the sebacinoid s.l. OTUs 848, Fig.3b, and 1014, Fig.3a) were 411 exclusively detected in the vegetative organs. 412 No significant difference was found in the distribution profiles of OrM fungal OTUs between 413 vegetative and reproductive structures (CAP, P>0.05, Table S3), but a significant difference was found again 414 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 17 between species, as well as for the interaction between species and organ type (CAP, P<0.001; Table S3), and 415 samples grouped according to orchid species in the NMDS space (3Dstress = 0.1251; linear fit, R2=0.81; Fig. 416 S9). For example, T. helicospora (OTU162), which occurred in most (≥60%) root, stem, leaf and capsule 417 samples in either S. vomeracea or N. ovata, was sporadic in S. spiralis_2, while the sebacinoid s.l. OTUs 497, 418 516 and 458, which were not retrieved at all, or only rarely, in S. spiralis_2 and S. vomeracea, respectively, 419 occurred in most N. ovata samples (Table S1). 420 421

422 423 The assemblages of fungi associated with internal orchid tissues have been investigated in several terrestrial 424 and epiphytic species, where most studies focused on the root -associated mycobiota because of the major 425 interest in OrM fungi (e.g. Bayman & Otero, 2006; Li et al., 2021; Ma et al., 2015; Rashmi et al., 2019; 426 Sarsaiya et al., 2020; Bhatti & Thakur, 2022). Investigations on the endophytic mycobiota associated with 427 other organs have instead been mostly restricted to economically important orchids, such as Dendrobium and 428 Vanilla species (e.g. Yuan et al., 2009; Chen et al., 2013, 2020; Parthibhan et al., 2017; Ma et al., 2022; 429 Mosquera -Espinosa & Rodríguez-Mina, 2022; Parthibhan et al., 2017; Sarsaiya et al., 2020; Zhu et al., 2022). 430 431 The mycobiota of vegetative plant organs include OrM fungi in all orchid species investigated 432 433 The primers used in this study retrieved large spectra of OTUs encompassing phylogenetically and ecologically 434 diverse fungi, as found in other orchids (Li et al., 2021; Ma et al., 2015; Rashmi et al., 2019; Bhatti & Thakur, 435 2022; Cevallos et al., 2022). The dominant OTUs in the three orchid species investigated included fungi in the 436 Ascomycota and Basidiomycota commonly reported in a wide range of hosts, such as species in Alternaria, 437 Aureobasidium, Cladosporium, Fusarium (Gibberella), Phoma sensu lato (Pyrenochaetopsis, Didymella), 438 Malassezia (Rashmi et al., 2019). Wood-decaying fungi (such as Bjerkandera adusta and other Polyporales, 439 Table S1)found as endophytes of several tree and herbaceous species (Brum et al., 2012; Martin et al., 2015; 440 Vaz et al., 2020; Faddetta et al., 2021; Parada et al., 2022 ), were also common . 441 As in other plants, the fungal assemblages were shaped by both the plant compartment and plant 442 species (Harrison & Griffin, 2020; Trivedi et al., 2020). Indeed, each orchid species harbored a distinctive 443 mycobiota and, within each orchid species, OTU profiles differed in distinct organs, suggesting they represent 444 dissimilar microhabitats. Despite these differences, however, several members of the orchid mycobiota were 445 shared by two or more organs, denoting either recruitment from a common environmental source, or the 446 capacity for active fungal growth through different plant parts. In all orchid species, roots and leaves of the 447 same plant exhibited greater overlap of taxa with stems than with each other, indicating the “intermediate” 448 nature of stems as a fungal habitat along the vertical axis from roots to aboveground organs (Amend et al., 449 2019; Bahram et al., 2022). Profiles of the dominant OTUs were especially similar in roots and stems, which 450 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 18 may be explained either by contamination of the lower parts of stems by rain splash from soil (Cooke & 451 Rayner, 1984) or by upwards fungal growth from belowground organs. 452 Interestingly, 33 OTUs identified as putative OrM fungi in the “rhizoctonia” complex were found not 453 only in roots but also in aerial organs of S. spiralis, S. vomeracea and N. ovata. Some of these OTUs (61.5%) 454 were detected in roots, leaves and stems of the same plant, indicating systemic colonization of the internal 455 orchid tissues. Like the general mycobiota, assemblages of these OrM fungi were shaped by both the orchid 456 species host and by the plant organ. 457 458 Role of rhizoctonia -like fungi in the orchid roots 459 460 As metabarcoding cannot test the actual mycorrhizal ability of the “rhizoctonia -like” fungi identified in the 461 roots, such studies may potentially overestimate mycorrhizal fungal diversity (Albornoz et al., 2021). 462 However, the occurrence of hyphal pelotons, the typical OrM fungal structures, was microscopically verified 463 in the roots of the three orchid species, and we assume that the peloton -forming fungi correspond to, or are 464 included in, the spectrum of the tulasnelloid, ceratobasidioid and sebacinoid s.l. OTUs identified in the roots. 465 The same OTUs clustered closely with sequences already reported as OrM in the same and in different orchid 466 species, and a Tulasnella helicospora strain was recently demonstrated to induce symbiotic seed germination 467 and protocorm colonization in vitro (Calevo et al., 2020). However, the possibility that some of the rhizoctonia-468 like fungi identified in our study do not form hyphal pelotons in the orchid roots cannot be ruled out entirely 469 and is discussed later. 470 The role of OrM fungi in the uptake and transfer of nutrients to adult orchids has been demonstrated 471 in vitro (Cameron et al., 2006, 2007, 2008; Read et al., 2024) and in nature (Gomes et al., 2023; Zahn et al., 472 2023, 2024). Nutrient transfer to the host plant is thought to occur across the plant -fungus interface that 473 surrounds the intracellular hyphal pelotons formed in OrM protocorms and roots (Perotto & Balestrini, 2024). 474 Several questions remain open on the nature of nutrients transferred to the orchid host by rhizoctonia-like OrM 475 fungi (Fochi et al., 2017; De Rose et al., 2023), but the role of these fungi in the supply of nutrients to the 476 orchid host and the site of nutrient transfer in mycorrhizal roots (i.e., the hyphal pelotons) have never been 477 questioned. 478 479 Potential role of rhizoctonia -like fungi as endophytes in orchid roots and aerial parts 480 481 Although the nutritional benefits for the host plant are generally ascribed to mycorrhizal fungi (Selosse et al., 482 2022), there is growing evidence that specialized structures such as those formed in mycorrhizal associations 483 are not essential for improved nutrient uptake and plant growth promotion, as some root -associated non-484 mycorrhizal fungal endophytes can aid nutrient absorption and nutrient allocation to different plant parts 485 through a range of mechanisms (Poveda et al., 2021; Sarkar et al., 2021; Verma et al., 2021; Watts et al., 486 2023). Some fungal endophytes can in fact increase the elemental composition (especially nitrogen and 487 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 19 phosphorous) in roots and shoots of inoculated plants, S. vermifera being the best studied example in non -488 orchid hosts (Verma et al., 2021). Therefore, we cannot exclude an additional role in plant nutrition by other 489 fungi among those identified in the orchid root mycobiota, or by rhizoctonia -like fungi that may possibly 490 colonize the orchid root without forming pelotons. 491 The type of interaction with the host plant and the role of these rhizoctonia -like OrM fungi in the 492 vegetative aerial plant parts (stems and leaves) is currently unknown, and we can only make speculations on 493 their functional role based on research on other fungal endophytes. The contribution of some fungal endophytes 494 which are not (or not exclusively) root endophytes to plant nutrient acquisition has been reported (Sarkar et 495 al., 2021). For instance, Epichloë species (Clavicipitaceae, Ascomycota) are endophytic symbionts of cool-496 season grasses (White, 1987). Whereas the Epichloë mycelium is found throughout the above-ground portions 497 of the grass plant, roots are typically not colonized (Rodriguez et al., 2009; Mosaddeghi et al., 2021). These 498 endophytes increase plant nutrient uptake by multiple mechanisms, including enhanced root proliferation by 499 indole-3-acetic acid production (Mosaddeghi et al ., 2021) and release of root exudates that facilitate 500 solubilization and absorption of some nutrients, such as P, K and Fe (Malinowski & Belesky, 2000; Soto -501 Barajas et al., 2016). 502 These findings indicate that connection with the soil environment is not an essential requirement for 503 aid in plant nutrient uptake by fungi associated with aerial plant parts (Kariman et al., 2018). For fungi 504 establishing systemic infection of orchids, like the rhizoctonia -like fungi identified in this study, the hyphal 505 connection between roots and aerial plant parts may represent an additional route for nutrient allocation in the 506 host plant. Moreover, non-nutritional benefits also accrue to endophyte-infected plant hosts. Colonization by 507 Epichloë spp. also confers plant tolerance to individual or combined abiotic (i.e., drought, salinity, mineral 508 and temperature extremes), as well as biotic stresses, including local and systemic resistance to pathogens 509 (Mosaddeghi et al., 2021). Intriguingly, some Ceratobasidium isolates from OrM orchid roots were effective 510 as biocontrol agents of Rhizoctonia solani (= Thanatephorus cucumeris), a pathogen that causes sheath blight 511 in rice (Mosquera-Espinosa et al., 2013). Other OrM isolates (one Ceratobasidium sp. and two Tulasnella spp. 512 isolates) showed some antagonistic in vitro abilities against Fusarium oxysporum f.sp. vanillae, the causative 513 agent of Fusarium wilt in the orchid Vanilla planifolia, but no biocontrol activity when inoculated in vivo 514 (Manrique-Barros et al., 2023). 515 Thus, although their role requires further investigations, OrM fungi have multiple opportunities, by 516 virtue of their systemic colonization of orchid tissues, to benefit their plant hosts at the adult stage. 517 518 Potential vertical transmission of OrM rhizoctonia -like fungi and benefits for the plant 519 520 Whatever their structural/functional relationship with the orchid tissues, the capacity of OrM fungi for systemic 521 colonization of the orchid organs has implications for the persistence and spread of these fungi in the 522 environment. The identification, in the capsules of the three orchid species, of a core mycobiota including 523 rhizoctonia-like OrM fungi also found in vegetative organs, indicates that these fungi could also colonize the 524 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 20 flower and the reproductive organs. For S. spiralis, it was possible to track putative OrM fungal OTUs also in 525 the seeds released from mature capsules. Similarly, a metabarcoding study by Wang et al. (2022) retrieved the 526 same tulasnelloid OTU in mature mycorrhizal roots and in capsules of wild Cymbidium goeringii plants in 527 China. Although more samples and species need to be investigated, these findings open the intriguing 528 possibility that some OrM fungi could be vertically transmitted. 529 Vertical transmission of systemic fungal endophytes from a mother plant to the new seedlings via the seeds 530 has been originally reported for Epichloë species in grasses (Liu et al., 2017), but it is likely more widespread 531 and includes many more species than previously thought (Hodgson et al ., 2014; Shahzad et al ., 2018; 532 Abdelfattah et al., 2021; Özkurt et al., 2020; Fort et al., 2021). Given the importance of OrM fungi in orchid 533 seed germination and recruitment, a strategy that allows the minute orchid seeds to travel with their own 534 symbiotic fungi would likely be evolutionarily successful, provided that the same fungi colonizing adult plants 535 can also promote seed germination and protocorm development. The spectrum of OrM fungi may in fact 536 change during protocorm development into adult plants in some orchid species (Rasmussen et al., 2015; Ventre 537 Lespiaucq et al., 2021), but most OrM fungi able to promote symbiotic seed germination were originally 538 isolated from adult plants (e,g, Fuji et al., 2020; McCormick et al., 2021; Těšitelová et al., 2022; Calevo & 539 Duffy, 2023; Fernández et al., 2023; Chamara et al., 2024; Freestone et al., 2024). Moreover, most results on 540 the local restriction of orchid distribution by OrM fungal abundance demonstrate that seed germination is 541 higher near adult orchids (see in McCormick et al., 2018), suggesting at least partially shared OrM fungi. 542 543 Possible ecological benefits of systemic endophytism for OrM fungi 544 545 For plants such as orchids, that rely heavily on symbiotic fungi throughout their life history stages, the 546 possibility to host important fungal taxa as systemic colonizers and to transmit them vertically to the offspring 547 via the seeds could be a successful strategy for survival and dispersal (Khan et al., 2015; Shahzad et al., 2018). 548 In return, OrM fungi could use their host plants for survival and persistence in the environment (Selosse, 2014; 549 Selosse & Martos, 2014; Oja et al., 2015; McCormick et al., 2016; Voyron et al., 2017). Studies by McCormick 550 et al. (2018) and Waud et al. (2016) showed that OrM fungi were abundant in small soil patches closely tied 551 to the locations of adult orchids, thus suggesting that adult orchid plants provide their OrM fungal symbionts 552 with a ‘refuge’. Under natural conditions, the prevalence of some rhizoctonia-like OrM fungi inside the host 553 plant and their undetectability in soils (Voyron et al., 2017; Egidi et al., 2017; Kaur et al., 2019; Hartvig et al., 554 2024) further suggest that some symbiotic fungi may even be unable to grow outside the host plant. For such 555 fungi, specialization on systemic biotrophism may lead to an ecological advantage, as plants may represent a 556 refugium from the stress and competition conditions associated with saprotrophic growth in soil (Cooke & 557 Rayner, 1984). 558 Endophytism appears to be often associated with environmental abiotic stress, like heat stress 559 (Matheny et al., 2018; Raudabaugh et al., 2020; Fox et al., 2022). In Mediterranean environments, like those 560 where our orchid species grow, foliar endophytism may be a possible adaptation to cope not only with heath, 561 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 21 but also with moisture limitation (Vaz et al., 2020). Thus, although questions remain open on the actual 562 capabilities of OrM fungi to grow in the soil as saprotrophs, endophytism may have been an alternative for 563 these fungi, especially in stressful environments such as the Mediterranean region. 564 565

566 Our results show that rhizoctonia-like fungi in the tulasnelloid, ceratobasidioid and sebacinoid s.l. clades not 567 only associate with orchid roots, where they likely form OrM, but they are also capable of endophytic growth 568 in the aerial tissues of their orchid hosts, including reproductive organs. Further investigations are required to 569 assess the colonization pattern, the viability and the functional role of rhizoctonia-like OrM fungi in the aerial 570 plant organs and in seeds, but these first results may suggest a specific strategy for survival and dispersal that 571 would benefit both the orchid host and the associated fungal partners. 572 An intriguing question is the evolution of the interactions between rhizoctonia-like OrM fungi and their orchid 573 host. As many rhizoctonia-like OrM fungi are also endophytes in non-orchid plants, it has been hypothesized 574 that extant OrM fungi were recruited among root endophytic fungi that colonized orchid ancestors (the ‘waiting 575 room’ hypothesis, Selosse et al., 2022). Our findings that OrM fungi can colonize at the same time both roots 576 and aerial plant organs of their extant orchid hosts make the scenario far more complex and expand the life 577 history traits and potential ecological roles of OrM fungi. 578 579 Acknowledgments 580 J.C. was funded by a European Commission Marie Skłodowska -Curie Global Fellowship (grant agreement 581 No. 101031324 “FORECAST – Quantifying the impact of climate change on orchid mycorrhizal symbiosis in 582 Mediterranean biodiversity hotspots”) . P.A. was supported by a PhD fellowship by MUR. Research was 583 supported by National funds PRIN 2017TP3SJL “Mediterranean orchids as a model system to quantify 584 intrinsic and extrinsic factors influencing distribution and conservation -oriented restoration” . 585 586 Competing interests 587 Authors declare no competing interests 588 589 Author contributions 590 All authors contributed to the study conception and design. Material preparation and data collection were 591 performed by J.C. and P.A. Data analysis was performed by all authors. The first draft of the manuscript was 592 written by S.P., J.C. and M.G., and all authors commented on previous versions of the manuscript, read and 593 approved the final manuscript . 594 595 Data availability 596 We confirm that, should the manuscript be accepted, the data supporting the results will be archived in Zenodo 597 under the DOI: 10.5281/zenodo.14223971. The ITS representative sequences of fungi from Supplementary 598 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 22 Table S1 were submitted to GenBank and recorded under the following string of accession numbers: 599 PQ644909 - PQ645014. 600 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 23

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It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 35 Zhao Z, Shao S, Liu N, Liu Q, Jacquemyn H, Xing X. 2021 . Extracellular enzyme activities and 964 carbon/nitrogen utilization in mycorrhizal fungi isolated from epiphytic and terrestrial orchids. Frontiers in 965 Microbiology 12: 787820. 966 Zhu MM, Chen HH, Si JP, Wu LS. 2022 . Effect of cultivation mode on bacterial and fungal communities 967 of Dendrobium catenatum . BMC Microbiology 22: 2635. 968 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 36 Tables and Figures 969 970 Figure captions 971 972 Figure 1. Floral morphology of the three target orchid species: (a) Spiranthes spiralis, (b) Serapias vomeracea, 973 and ( c) Neottia ovata . Each panel illustrates the distinct floral characteristics unique to each species, 974 highlighting their morphological diversity. 975 976 Figure 2. Heatmap and hierarchical clustering of OrM OTU frequency distribution across plant species and 977 organs. The heatmap displays the frequency percentage (0 -100%) of different OTUs in stem, leaf, and root 978 samples from the target orchid species ( Spiranthes spiralis_1, Spiranthes spiralis_2, Serapias vomeracea, 979 Neottia ovata). The dendrogram on the right shows the hierarchical clustering of samples based on their OTU 980 composition similarity. Color intensity from white to dark blue represents increasing OTU frequency. 981 982 Figure 3 . Circular diagrams illustrating the distribution of OTUs (Operational Taxonomic Units) across 983 different plant organs for three species: ( a) Spiranthes spiralis , (b) Serapias vomeracea , (c) Neottia ovata. 984 Each diagram shows connections between OTUs and plant organs (root, leaf, stem, capsule, seed), with color-985 coded segments representing different organs. The width of the connections indicates the abundance of OTUs 986 in each organ, highlighting the diversity and distribution patterns within each species. Blue arches indicate 987 ceratobasidioid OTUs, green arches tulasnelloid OTUs, while yellow arches indicate sebacinoid s.l. OTUs. 988 989 Figure 4. Non-metric multidimensional scaling (NMDS) plot of plant organ samples based on species. This 990 NMDS plot visualizes the relationships among different plant organs (leaf, root, stem) based on OTUs 991 similarities across four species: Neottia ovata, Serapias vomeracea, Spiranthes spiralis _1, and Spiranthes 992 spiralis_2. The axes represent NMDS1 and NMDS2 dimensions, with a stress value of 0.129818 indicating a 993 good fit for the ordination. Data points are shaped according to organ type and colored by species. 994 995 Supplementary material 996 997 Table S1. Taxonomic affiliation (according to NCBI) of the OTUs occurring in ≥80% plants in at least one 998 orchid species and the 33 rhizoctonia-like OTUs. Occurrences in the orchid vegetative organs (percentage of 999 plants hosting in each vegetative organ in each orchid species) are also reported. Spir1= Spiranthes spiralis_1; 1000 Spir2= S. spiralis _2; Ser= Serapias vomeacea ; Neo= Neottia ovata . 1001 1002 Table S2. PERMANOVA results assessing the effects of species, organ, and their interaction on the Bray -1003 Curtis and Jaccard dissimilarity matrixes of the OrM fungi dataset. The analysis was conducted using 9999 1004 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 37 permutations to determine the significance of each term, revealing that species, organ, and their interaction 1005 significantly influence the community composition, as indicated by the F -values and R² values. 1006 1007 Table S3. Summary of Constrained Analysis of Principal Coordinates (CAP) results comparing the effects of 1008 species identity, organ type (vegetative or reproductive) , and their interaction on OrM fungal community 1009 composition. The table shows the F-statistic, R² (proportion of variance explained), p-values, and significance 1010 levels for each factor and their interaction. Analyses were performed using both Bray -Curtis and Jaccard 1011 distance metrics. Significance levels are indicated as: *** (p < 0.001), ** (p < 0.01), * (p < 0.05), ns (not 1012 significant). The results show that species identity and the interaction between species and organ type have a 1013 highly significant effect on OrM fungal community composition (p < 0.001) with both distance metrics . 1014 1015 Figure S1. Venn diagrams showing the number of shared and organ-specific fungal OTUs in the roots, stems 1016 and leaves of the same plant for each of the three orchid species: Spiranthes spiralis, Serapias vomeracea and 1017 Neottia ovata . For S. spiralis , plants were sampled in two consecutive years. 1018 1019 Figure S2. Principal Component Analysis (PCA) of fungal communities associated with different orchid 1020 organs based on Jaccard (left) and Bray -Curtis (right) dissimilarity matrices. The analysis includes fungal 1021 OTUs from three orchid taxa (Neottia, Serapias, and two Spiranthes group samples) and three plant organs 1022 (leaf, root, and stem). Points are shaped according to the organ type (circles = leaf, triangles = root, crosses = 1023 stem) and colored by orchid taxa. The percentage of variance explained by each principal component is shown 1024 on the axes. PCoA1 explains 15.33% and 18.25% of the total variance for Jaccard and Bray-Curtis analyses 1025 respectively. 1026 1027 Figure S3. Heatmap and hierarchical clustering of the dominant non-OrM fungal OTUs across plant species 1028 and organs. The heatmap displays OTU frequency percentages (0-100%) in different plant organs (stem, root, 1029 leaf) of the target orchids: Spiranthes spiralis_1 (Spir1), Spiranthes spiralis_2 (Spir2), Neottia ovata (Neo), 1030 Serapias vomeracea (Ser). Hierarchical clustering (dendrogram) shows sample relationships. Color intensity 1031 from white to dark blue represents increasing OTU frequency. 1032 1033 Figure S4 . Maximum likelihood tree obtained from the ITS2 sequence alignment of tulasnelloid fungi. 1034 Multicalvula vernalis was used as outgroup. Bootstrap support values above 70% (1000 maximum likelihood 1035 replicates) are reported. OTUs found in this study are indicated in bald, sequences retrieved in previous studies 1036 from the same orchid species are in red. 1037 1038 Figure S5. Maximum likelihood tree obtained from the ITS2 sequence alignment of ceratobasidioid fungi. 1039 Tricholoma portentosum and Laccaria bicolor were used as outgroup taxa. Bootstrap support values above 1040 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint 38 70% (1000 maximum likelihood replicates) are reported. OTUs found in this study are indicated in bald, 1041 sequences retrieved in previous studies from the same orchid species are in red. 1042 1043 Figure S6 . Maximum likelihood tree obtained from the ITS2 sequence alignment of fungi assigned to 1044 Sebacinales. Tremiscus helvelloides was used as an outgroup taxon. Bootstrap support values above 70% (1000 1045 maximum likelihood replicates) are reported. OTUs found in this study are indicated in bald, sequences 1046 retrieved in previous studies from the same orchid species are in red. 1047 1048 Figure S7. Venn diagrams showing the number of tulasnelloid, ceratobasidioid and sebacinoid s.l. OTUs in 1049 the roots, stems and leaves of the same plant for each of the three orchid species: Spiranthes spiralis, Serapias 1050 vomeracea and Neottia ovata. The list of OTUs shared between different organs is provided. For S. spiralis, 1051 plants were sampled in two consecutive years. 1052 1053 Figure S8. The larger pie charts depict the share of occurrences of the 33 rhizoctonia OTUs in either all (RSL), 1054 two (RS, roots&stem; RL, roots&leaves and LS, leaves&stem), or a single vegetative organ (R, roots; S, stem; 1055 L, leaves) in the same plant for each orchid species. The smaller charts summarize the share of total occurrences 1056 in roots, stems and leaves. 1057 1058 Figure S9. Non-metric multidimensional scaling (NMDS) plot illustrating the relationships among different 1059 plant organs and species based on their characteristics. The axes represent NMDS1 and NMDS2 dimensions. 1060 Data points are shaped according to organ type (capsule, leaf, root, seed, stem) and colored by species (Neottia 1061 ovata, Serapias vomeracea, Spiranthes spiralis_2). Ellipses indicate clustering patterns, with a stress value of 1062 0.125118, suggesting a good fit for the ordination. 1063 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 13, 2025. ; https://doi.org/10.1101/2025.01.08.631981doi: bioRxiv preprint