Three-year pot culture of Epipactis helleborine reveals autotrophic survival, without mycorrhizal networks, in a mixotrophic species

Abstract

Some mixotrophic plants from temperate forests use the mycorrhizal fungi colonizing their roots as a carbon source to supplement their photosynthesis. These fungi are also mycorrhizal on surrounding trees, from which they transfer carbon to mixotrophic plants. These plants are thus reputed difficult to transplant, even when their protection requires it. Here, we take profit of a successful ex situ pot cultivation over 1 to 3 years of the mixotrophic orchid Epipacis helleborine to investigate its mycorrhizal and nutrition status. Firstly, compared with surrounding autotrophic plants, it did not display the higher N content and higher isotopic (13 C and 15 N) abundance that normally feature mixotrophic orchids because they incorporate N-, 13 C-, and 15 N-rich fungal biomass. Second, fungal barcoding by next-generation sequencing revealed that the proportion of ectomycorrhizal fungi (expressed as percentage of the total number of either reads or operational taxonomic units) was unusually low compared with E. helleborine growing in situ: instead, we found a high percentage of rhizoctonias, the usual mycorrhizal partners of autotrophic orchids. Altogether, this supports autotrophic survival. Added to the recently published evidence that plastid genomes of mixotrophic orchids have intact photosynthetic genes, this suggests that at least some of them have abilities for autotrophy. This adds to the ecological plasticity of mixotrophic plants, and may allow some reversion to autotrophy in their evolution.

How Mycorrhizal Associations Influence Orchid Distribution and Population Dynamics

“…Preliminary research suggests that the dominant OMF and ONF in fungal communities reconstructed by cultured orchid individuals are different from those seen in wild populations. Overall, fungi belonging to the groups Atractiellales, Auriculariales, Ceratobasidiaceae, and Fusarium tended to increase, while the abundance of Tulasnellaceae and Pyronemataceae tended to decrease (Downing et al, 2017;Qin et al, 2019;May et al, 2020), as a consequence of transplantation, and the extent of such changes may depend on the time scales of culture or transplantation. This indicates low survival rates of cultured orchids, which may be caused by the loss of some key OMF (e.g., Tulasnellaceae fungi) or the restricted construction of new mycorrhizal communities.…”

“…Studies have shown that the use of peat-based, a mixture of coconut shells, bark shavings, soil from the original habitat, or a mixture of pinecone scales, mosses, and humus, can induce symbiosis between transplanted orchid individuals or asymbiotically cultured orchid individuals grown ex situ and Tulasnellaceae fungi (Han et al, 2016;Kaur et al, 2018;Qin et al, 2019). However, no Tulasnella symbiotic with orchid individuals were obtained when transplanted in original habitat soil or sawdust (May et al, 2020;Li et al, unpublished data). Preliminary research suggests that the dominant OMF and ONF in fungal communities reconstructed by cultured orchid individuals are different from those seen in wild populations.…”

Progress and Prospects of Mycorrhizal Fungal Diversity in Orchids

“…Further, Selosse et al (2004) observed Ascomycota from the genus Tuber, based on molecular identification, transmission electron microscope, and immunolabeling in Epipactis microphylla. Ascomycota were sporadically reported, e.g., as rare OMF in MX orchids, such as Limodorum abortivum and Epipactis helleborine (Girlanda et al, 2006;May et al, 2020;Xing et al, 2020), and in South-African orchids (Waterman et al, 2011). Some AT orchids are associated with Atractiellales (Pucciniomycotina; Kottke et al, 2010;Cevallos et al, 2018b;Qin et al, 2019;Xing et al, 2019) and saprotrophic fungi from Mycenaceae or ECM fungi from Russulaceae, Peziza, and Inocybe (Waterman et al, 2011;Zhang et al, 2012;Esposito et al, 2016;Waud et al, 2017;Xing et al, 2020).…”

“…Some AT orchids are associated with Atractiellales (Pucciniomycotina; Kottke et al, 2010;Cevallos et al, 2018b;Qin et al, 2019;Xing et al, 2019) and saprotrophic fungi from Mycenaceae or ECM fungi from Russulaceae, Peziza, and Inocybe (Waterman et al, 2011;Zhang et al, 2012;Esposito et al, 2016;Waud et al, 2017;Xing et al, 2020). Additionally, a few photosynthetic orchids associate with saprotrophic Auriculariales, Psathyrellaceae, Tricharina, Clavulina, Armillaria, Marasmius, and Scleroderma fungi belonging to the common ECM fungal taxa (Waterman et al, 2011;Yagame et al, 2013;Jacquemyn et al, 2016b;González-Chávez et al, 2018;Qin et al, 2019;May et al, 2020;Salazar et al, 2020). The presence of such fungi, likely in minor amounts, was greatly enhanced by the use of HTS and the reporting of all the diversity found, without a priori screening (Selosse et al, 2010).…”

“…However, there are a few specific MX orchids, such as P. minor and L. abortivum that specifically associate with ECM Ceratobasidium fungi and Russula delica, respectively (Girlanda et al, 2006;Yagame et al, 2012). In a Pinus thunbergii plantation, the MX E. helleborine displayed abundant Wilcoxina fungi, but whether this is an OMF remains unclear (Suetsugu et al, 2017), while this species is normally poorly specific (May et al, 2020).…”