Can orchid mycorrhizal fungi be persistently harbored by the plant host?

The environmental distribution of non-obligate orchid mycorrhizal (OM) symbionts belonging to the ‘rhizoctonia’ complex remains elusive. Some of these fungi, indeed, are undetectable in soil outside the host rhizosphere. A manipulation experiment was performed to assess the importance of neighbouring non-orchid plants and soil as possible reservoirs of OM fungi for Spiranthes spiralis, a widespread photosynthetic European terrestrial orchid species. Fungi of S. spiralis roots were identified by DNA metabarcoding before and 4 months after the removal of the surrounding vegetation and soil. Although such a treatment significantly affected fungal colonization of newly-formed orchid roots, most OM fungi were consistently associated with the host roots. Frequency patterns in differently aged roots suggest that these fungi colonize new orchid roots from either older roots or other parts of the same plant, which may thus represent an environmental source for the subsequent establishment of the OM symbiosis.     

Evolution of host breadth in broad interactions: mycorrhizal specificity in East Asian and North American rattlesnake plantains (Goodyera spp.) and their fungal hosts

Host breadth is often assumed to have no evolutionary significance in broad interactions because of the lack of cophylogenetic patterns between interacting species. Nonetheless, the breadth and suite of hosts utilized by one species may have adaptive value, particularly if it underlies a common ecological niche among hosts. Here, we present a preliminary assessment of the evolution of mycorrhizal specificity in 12 closely related orchid species (genera Goodyera and Hetaeria) using DNA‐based methods. We mapped specificity onto a plant phylogeny that we estimated to infer the evolutionary history of the mycorrhiza from the plant perspective, and hypothesized that phylogeny would explain a significant portion of the variance in specificity of plants on their host fungi. Sampled plants overwhelmingly associated with genus Ceratobasidium, but also occasionally with some ascomycetes. Ancestral mycorrhizal specificity was narrow in the orchids, and broadened rarely as Goodyera speciated. Statistical tests of phylogenetic inertia suggested some support for specificity varying with increasing phylogenetic distance, though only when the phylogenetic distance between suites of fungi interacting with each plant taxon were taken into account. These patterns suggest a role for phylogenetic conservatism in maintaining suits of fungal hosts among plants. We stress the evolutionary importance of host breadth in these organisms, and suggest that even generalists are likely to be constrained evolutionarily to maintaining associations with their symbionts.     

Identity and specificity of the fungi forming mycorrhizas with the rare mycoheterotrophic orchid Rhizanthella gardneri

Fully subterranean Rhizanthella gardneri (Orchidaceae) is obligately mycoheterotrophic meaning it is nutritionally dependent on the fungus it forms mycorrhizas with. Furthermore, R. gardneri purportedly participates in a nutrient sharing tripartite relationship where its mycorrhizal fungus simultaneously forms ectomycorrhizas with species of Melaleuca uncinata s.l. Although the mycorrhizal fungus of R. gardneri has been morphologically identified as Thanatephorus gardneri (from a single isolate), this identification has been recently questioned. We sought to clarify the identification of the mycorrhizal fungus of R. gardneri, using molecular methods, and to identify how specific its mycorrhizal relationship is. Fungal isolates taken from all sites where R. gardneri is known to occur shared almost identical ribosomal DNA (rDNA) sequences. The fungal isolate rDNA most closely matched that of other Ceratobasidiales species, particularly within the Ceratobasidium genus. However, interpretation of results was difficult as we found two distinct ITS sequences within all mycorrhizal fungal isolates of R. gardneri that we assessed. All mycorrhizal fungal isolates of R. gardneri readily formed ectomycorrhizas with a range of M. uncinata s.l. species. Consequently, it is likely that R. gardneri can form a nutrient sharing tripartite relationship where R. gardneri is connected to autotrophic M. uncinata s.l. by a common mycorrhizal fungus. These findings have implications for better understanding R. gardneri distribution, evolution and the ecological significance of its mycorrhizal fungus, particularly in relation to nutrient acquisition.     

Arbuscular, ecto-related, orchid mycorrhizas—three independent structural lineages towards mycoheterotrophy: implications for classification?

The classification of mycorrhizas in seven equally ranked types glosses over differences and similarities and, in particular, does not acknowledge the structural diversity of arbuscular mycorrhizas. This article emphasizes the parallel continua of ecto-related mycorrhizas and arbuscular mycorrhizas, exemplified within Ericaceae and Gentianales, respectively, as well as the proprietary development of orchid mycorrhizas, all three of which have independently developed mycoheterotrophic plants. A hierarchical classification according to structural similarities is suggested.     

Identification of mycobionts in an achlorophyllous orchid,Cremastra aphylla (Orchidaceae), based on molecular analysis and basidioma morphology

Mycorrhizal fungi were isolated and cultured from rhizomes of mycoheterotrophic Cremastra aphylla (Orchidaceae) plants collected in 3 sites across Japan. In total, 5 Cr. aphylla individuals were collected, and 10 fungal isolates were obtained. Sequence analysis of the internal transcribed spacer regions (ITS) of nuclear ribosomal DNA from the fungal samples revealed that all isolates belonged to the genus Coprinellus in the family Psathyrellaceae. All isolates from each site were of the same phylotype. In total, 3 ITS phylotypes were detected. One of the isolates produced fruiting bodies and was identified as Co. domesticus on the basis of macro- and microscopic characteristics of the basidiomata and ITS sequence data. In this study, the sharing of saprobic Psathyrellaceae fungus by the mycoheterotrophic and leafy Cremastra species was newly confirmed.     

The effect of carbohydrate on the development of a Cattleya hybrid in association with its mycorrhizal fungus

To investigate beneficial effects of mycorrhizal fungi to advanced leafy orchids, growth studies on the development of symbiotic seedlings of the orchid Cattleya (aclandiae x schoeffeldiana) x aclandiae were conducted in vitro over a period of 18 months using split plates with minerals and carbohydrates on one side and water agar on the other. Mycorrhizal infection and shoot and root growth of seedlings on the nutrient side were compared to growth on the water agar side with nutrient uptake by the orchid only possible via external mycorrhizal hyphae. Seed germination was followed by mycorrhizal infection and rapid development of protocorms on both nutrient and non-nutrient sides of the plates. With 0.5% starch, development of protocorms was sustained for a least 12 weeks, compared to only 6 weeks with 0.1% starch. Advanced protocorms with two small leaves and a smoll root were transferred at week 22 to new fungal plates. When harvested at week 43, plantlets on 0.5% starch (both nutrient and water agar sides) had 2.7 times the dry weight of plantlets on 0.1% starch. Shoot-root ratios were higher on the lower level of carbon. In all plantlets, mycorrhizal infection involved less than 5% of the root length. With zero, 0.1% or 0.5% starch, the roots were re-infected on transfer to fresh fungal plates but young roots that developed following the transfer stayed free of infection, Plantlets on 0.5% starch (nutrient and water agar side) after 18 months had longer roots than plantlets grown in the absence of starch or on 0.1% starch. Shoots were small but significantly larger on the nutrient side than on the water agar side, independent of the carbohydrate level. The shoot-root ratio was highest on the nutrient side with no starch present. In this latter case, plantlet development was steady but plantlets on the non-nutrient side developed slowly; thus there was little evidence of nutrient translocation by the mycorrhizal fungus from the nutrient to the non-nutrient side in the absence of carbohydrates. Mycorrhizal infection is discussed as a mechanism for heterotrophic carbon assimilation. In advanced leafy orchids of Cattleya, external carbon resulted in increased root growth, decreased shoot/root ratio and sometimes yellowish-green plantlets.     

Three new species in the genus Tulasnella isolated from orchid mycorrhiza of Spiranthes sinensis var. amoena (Orchidaceae)

We isolated Tulasnella spp. from Spiranthes sinensis var. amoena, a Japanese native winter green terrestrial orchid collected in Tsukuba City, Ibaraki Pref., Japan. These isolates were classified into four morphotypes according to morphological characters, i.e., shape of monilioid cells and branch type of monilioid cell chains, while they were separated into five clades by molecular phylogenetic analysis based on the rDNA 5.8S and D1/D2 regions. The four morphotypes and five clades were correlated, and four morpho-phylogenetic groups were identified. Thus, Tulasnella deliquescens including two phylogenetic subgroups and three new species, Tulasnella dendritica sp. nov., Tulasnella ellipsoidea sp. nov., and Tulasnella cumulopuntioides sp. nov., were recognized in this study. In this study, the monilioid cell chain morphology is newly defined, and is suggested as a useful taxonomic characteristic in the asexual stage of Tulasnella spp.     

Evolution of nutritional modes of Ceratobasidiaceae (Cantharellales,Basidiomycota) as revealed from publicly available ITS sequences

Fungi from the Ceratobasidiaceae family have important ecological roles as pathogens, saprotrophs, non-mycorrhizal endophytes, orchid mycorrhizal and ectomycorrhizal symbionts, but little is known about the distribution and evolution of these nutritional modes. All public ITS sequences of Ceratobasidiaceae were downloaded from databases, annotated with ecological and taxonomic metadata, and tested for the non-random phylogenetic distribution of nutritional modes. Phylogenetic analysis revealed six main clades within Ceratobasidiaceae and a poor correlation between molecular phylogeny and morphological–cytological characters traditionally used for taxonomy. Sequences derived from soil (representing putative saprotrophs) and orchid mycorrhiza clustered together, but remained distinct from pathogens. All nutritional modes were phylogenetically conserved in the Ceratobasidiaceae based on at least one index. Our analyses suggest that in general, autotrophic orchids form root symbiosis with available Ceratobasidiaceae isolates in soil. Ectomycorrhiza-forming capability has evolved twice within the Ceratobasidiaceae and it had a strong influence on the evolution of mycoheterotrophy and host specificity in certain orchid taxa.