Metabolomic adjustments in the orchid mycorrhizal fungus Tulasnella calospora during symbiosis with Serapias vomeracea.

All orchids rely on mycorrhizal fungi for organic carbon, at least during early development. In fact, orchid seed germination leads to the formation of a protocorm, a heterotrophic postembryonic structure colonized by intracellular fungal coils, thought to be the site of nutrient transfer. The molecular mechanisms underlying mycorrhizal interactions and metabolic changes induced by this symbiosis in both partners remain mostly unknown. We studied plant-fungus interactions in the mycorrhizal association between the Mediterranean orchid Serapias vomeracea and the basidiomycete Tulasnella calospora using nontargeted metabolomics. Plant and fungal metabolomes obtained from symbiotic structures were compared with those obtained under asymbiotic conditions. Symbiosis induced substantial metabolomic alterations in both partners. In particular, structural and signaling lipid compounds increased markedly in the external fungal mycelium growing near the symbiotic protocorms, whereas chito-oligosaccharides were identified uniquely in symbiotic protocorms. This work represents the first description of metabolic changes occurring in orchid mycorrhiza. These results - combined with previous transcriptomic data - provide novel insights on the mechanisms underlying the orchid mycorrhizal association and open intriguing questions on the role of fungal lipids in this symbiosis.

Fine-scale spatial distribution of orchid mycorrhizal fungi in the soil of host-rich grasslands.

Mycorrhizal fungi are essential for the survival of orchid seedlings under natural conditions. The distribution of these fungi in soil can constrain the establishment and resulting spatial arrangement of orchids at the local scale, but the actual extent of occurrence and spatial patterns of orchid mycorrhizal (OrM) fungi in soil remain largely unknown. We addressed the fine-scale spatial distribution of OrM fungi in two orchid-rich Mediterranean grasslands by means of high-throughput sequencing of fungal ITS2 amplicons, obtained from soil samples collected either directly beneath or at a distance from adult Anacamptis morio and Ophrys sphegodes plants. Like ectomycorrhizal and arbuscular mycobionts, OrM fungi (tulasnelloid, ceratobasidioid, sebacinoid and pezizoid fungi) exhibited significant horizontal spatial autocorrelation in soil. However, OrM fungal read numbers did not correlate with distance from adult orchid plants, and several of these fungi were extremely sporadic or undetected even in the soil samples containing the orchid roots. Orchid mycorrhizal 'rhizoctonias' are commonly regarded as unspecialized saprotrophs. The sporadic occurrence of mycobionts of grassland orchids in host-rich stands questions the view of these mycorrhizal fungi as capable of sustained growth in soil.

Cell-specific expression of plant nutrient transporter genes in orchid mycorrhizae.

Orchid mycorrhizal protocorms and roots are heterogeneous structures composed of different plant cell-types, where cells colonized by intracellular fungal coils (the pelotons) are close to non-colonized plant cells. Moreover, the fungal coils undergo rapid turnover inside the colonized cells, so that plant cells containing coils at different developmental stages can be observed in the same tissue section. Here, we have investigated by laser microdissection (LMD) the localization of specific plant gene transcripts in different cell-type populations collected from mycorrhizal protocorms and roots of the Mediterranean orchid Serapias vomeracea colonized by Tulasnella calospora. RNAs extracted from the different cell-type populations have been used to study plant gene expression, focusing on genes potentially involved in N uptake and transport and previously identified as up-regulated in symbiotic protocorms. Results clearly showed that some plant N transporters are differentially expressed in cells containing fungal coils at different developmental stages, as well as in non-colonized cells, and allowed the identification of new functional markers associated to coil-containing cells.