While orchids germinate thanks to carbon from their symbiotic fungi, variable carbon exchanges exist between adult orchids and their mycorrhizal fungi. Although some truly autotrophic orchids reward their fungi with carbon at adulthood, some species remain achlorophyllous and fully dependent on fungal carbon (mycoheterotrophy). Others are photosynthetic but also import fungal carbon: The so-called mixotrophic (MX) orchids rely on fungi of diverse taxonomy and ecology. Here, we classify MX nutrition of orchids into three types. Type I mixotrophy associates with diverse Asco- and Basidiomycota that are either saprotrophic or ectomycorrhizal, entailing enrichment of the orchids in 2H, 13C, and 15N. The two other types associate with rhizoctonias, a polyphyletic assemblage of Basidiomycotas that is ancestrally mycorrhizal in orchids. Type II mixotrophy associates with rhizoctonias that secondarily evolved into saprotrophic or ectomycorrhizal ecology, and thus enrich the orchid in 2H, 13C, and 15N. Type III mixotrophy, which remains debated, associates with rhizoctonias that have retained their ancestral lifestyle, that is saprotrophic and/or endophytic in nonorchids, and only entail orchid enrichment in 2H and 15N. Based on a case study of achlorophyllous variants in Mediterranean Ophrys and on published data, we discuss the distinct nature and research perspectives of type III mixotrophy.

• Keywords
• isotopic enrichment, mycoheterotrophy, mycorrhiza, rhizoctonia, saprotrophy,
• MeSH
• Basidiomycota physiology   MeSH
• Phylogeny MeSH
• Heterotrophic Processes MeSH
• Mycorrhizae physiology   MeSH
• Orchidaceae * microbiology   physiology   MeSH
• Symbiosis MeSH
• Carbon metabolism   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Carbon MeSH

Many orchid species are endangered due to anthropogenic pressures such as habitat destruction and overharvesting, meanwhile, all orchids rely on orchid mycorrhizal fungi (OMF) for seed germination and seedling growth. Therefore, a better understanding of this intimate association is crucial for orchid conservation. Isolation and identification of OMF remain challenging as many fungi are unculturable. In our study, we tested the efficiency of both culture-dependent and culture-independent methods to describe OMF diversity in multiple temperate orchids and assessed any phylogenetic patterns in cultivability. The culture-dependent method involved the cultivation and identification of single pelotons (intracellular hyphal coils), while the culture-independent method used next-generation sequencing (NGS) to identify root-associated fungal communities. We found that most orchid species were associated with multiple fungi, and the orchid host had a greater impact than locality on the variability in fungal communities. The culture-independent method revealed greater fungal diversity than the culture-dependent one, but despite the lower detection, the isolated fungal strains were the most abundant OMF in adult roots. Additionally, the abundance of NGS reads of cultured OTUs was correlated with the extent of mycorrhizal root colonization in orchid plants. Finally, this limited-scale study tentatively suggests that the cultivability character of OMF may be randomly distributed along the phylogenetic trees of the rhizoctonian families.

• Keywords
• Ceratobasidiaceae, Orchidaceae, Serendipitaceae, Tulasnellaceae, culture-independent and -dependent methods, fungal phylogeny, metabarcoding, mycorrhizal fungi,
• Publication type
• Journal Article MeSH

Orchid mycorrhizal fungi (OMF) from the rhizoctonia aggregate are generally considered to be soil saprotrophs, but their ability to utilize various nutrient sources has been studied in a limited number of isolates cultivated predominantly in liquid media, although rhizoctonia typically grow on the surface of solid substrates. Nine isolates representing the key OMF families (Ceratobasidiaceae, Tulasnellaceae and Serendipitaceae), sampled in Southern France and the Czech Republic, were tested for their ability to utilize carbon (C), nitrogen (N) and phosphorus (P) sources in vitro in both liquid and solid media. The isolates showed significant inter- and intra-familiar variability in nutrient utilization, most notably in N sources. Isolates produced generally larger amounts of dry biomass on solid medium than in liquid one, but some isolates showed no or limited biomass production on solid medium with particular nutrient sources. The largest amount of biomass was produced by isolates from the family Ceratobasidiaceae on most sources in both medium types. The biomass production of Tulasnellaceae isolates was affected by their phylogenetic relatedness on all sources and medium types. The ability of isolates to utilize particular nutrients in a liquid medium but not a solid one should be considered when optimizing solid media for symbiotic orchid seed germination and in understanding of OMF functional traits under in situ conditions.

• Keywords
• Ceratobasidiaceae, Orchidaceae, Serendipitaceae, Tulasnellaceae, liquid medium, nutrient utilization, rhizoctonia, solid medium,
• Publication type
• Journal Article MeSH

BACKGROUND: As in most land plants, the roots of orchids (Orchidaceae) associate with soil fungi. Recent studies have highlighted the diversity of the fungal partners involved, mostly within Basidiomycotas. The association with a polyphyletic group of fungi collectively called rhizoctonias (Ceratobasidiaceae, Tulasnellaceae and Serendipitaceae) is the most frequent. Yet, several orchid species target other fungal taxa that differ from rhizoctonias by their phylogenetic position and/or ecological traits related to their nutrition out of the orchid roots (e.g. soil saprobic or ectomycorrhizal fungi). We offer an evolutionary framework for these symbiotic associations. SCOPE: Our view is based on the 'Waiting Room Hypothesis', an evolutionary scenario stating that mycorrhizal fungi of land flora were recruited from ancestors that initially colonized roots as endophytes. Endophytes biotrophically colonize tissues in a diffuse way, contrasting with mycorrhizae by the absence of morphological differentiation and of contribution to the plant's nutrition. The association with rhizoctonias is probably the ancestral symbiosis that persists in most extant orchids, while during orchid evolution numerous secondary transitions occurred to other fungal taxa. We suggest that both the rhizoctonia partners and the secondarily acquired ones are from fungal taxa that have broad endophytic ability, as exemplified in non-orchid roots. We review evidence that endophytism in non-orchid plants is the current ecology of many rhizoctonias, which suggests that their ancestors may have been endophytic in orchid ancestors. This also applies to the non-rhizoctonia fungi that were secondarily recruited by several orchid lineages as mycorrhizal partners. Indeed, from our review of the published literature, they are often detected, probably as endophytes, in extant rhizoctonia-associated orchids. CONCLUSION: The orchid family offers one of the best documented examples of the 'Waiting Room Hypothesis': their mycorrhizal symbioses support the idea that extant mycorrhizal fungi have been recruited among endophytic fungi that colonized orchid ancestors.

• Keywords
• Ectomycorrhizal fungi, endophytism, mixotrophy, mycoheterotrophy, rhizoctonias, saprobic fungi,
• MeSH
• Waiting Rooms MeSH
• Endophytes MeSH
• Phylogeny MeSH
• Mycorrhizae * MeSH
• Orchidaceae * microbiology   MeSH
• Symbiosis MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

Many orchid species are threatened, while some disappear from their natural habitats without obvious reasons. Eutrophication has been suggested as a possible factor and nitrate, which is able to suppress non-symbiotic orchid seed germination even at very low concentrations, and could pose a serious threat for natural orchid populations. Early ontogenesis of all orchids entirely depends on orchid mycorrhizal symbiosis, and at this initial mycoheterotrophic stage, many terrestrial green orchids associate with polyphyletic fungal symbionts (i.e., mycobionts), collectively called "rhizoctonias." We asked whether these fungi might also have some non-nutritional roles, i.e., whether they might confer resistance to eutrophication. To test this hypothesis, we co-cultivated seeds of the terrestrial orchid Dactylorhiza majalis with five rhizoctonias (two Tulasnella, two Ceratobasidium and one Serendipita isolate) at various ecologically meaningful nitrate concentrations (0 to 100 mg/L). With the exception of one Tulasnella isolate, all mycobionts supported the growth of protocorms and formed orchid mycorrhiza, i.e., intracellular hyphal pelotons, in the protocorms. Nitrate suppressed asymbiotic, as well as symbiotic, seed germination in all but one fungal treatment; the seeds co-cultivated with one of the Ceratobasidium isolates were indeed insensitive to nitrate. We conclude that nitrates also negatively affect symbiotic orchid germination, depending on the available compatible mycobionts. Thus, eutrophication with nitrate may decrease the number of orchid mycobionts capable of supporting seed germination.

• Keywords
• Nitrate, Orchid mycorrhiza, Orchid seed germination, Protocorm development, Symbiotic fungi, Terrestrial green orchids,
• MeSH
• Nitrates MeSH
• Germination MeSH
• Mycorrhizae * MeSH
• Orchidaceae * MeSH
• Seeds MeSH
• Symbiosis MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Nitrates MeSH