Michele Rodda

Photosynthetic Mediterranean meadow orchids feature partial mycoheterotrophy and specific mycorrhizal associations

PREMISE OF THE STUDY We investigated whether four widespread, photosynthetic Mediterranean meadow orchids (Ophrys fuciflora, Anacamptis laxiflora, Orchis purpurea, and Serapias vomeracea) had either nutritional dependency on mycobionts or mycorrhizal fungal specificity. Nonphotosynthetic orchids generally engage in highly specific interactions with fungal symbionts that provide them with organic carbon. By contrast, fully photosynthetic orchids in sunny, meadow habitats have been considered to lack mycorrhizal specificity. METHODS We performed both culture-dependent and culture-independent ITS sequence analysis to identify fungi from orchid roots. By analyzing stable isotope ((13)C and (15)N) natural abundances, we also determined the degree of autotrophy and mycoheterotrophy in the four orchid species. KEY RESULTS Phylogenetic and multivariate comparisons indicated that Or. purpurea and Oph. fuciflora featured lower fungal diversity and more specific mycobiont spectra than A. laxiflora and S. vomeracea. All orchid species were significantly enriched in (15)N compared with neighboring non-orchid plants. Orchis purpurea had the most pronounced N gain from fungi and differed from the other orchids in also obtaining C from fungi. CONCLUSIONS These results indicated that even in sunny Mediterranean meadows, orchids may be mycoheterotrophic, with correlated mycorrhizal fungal specificity.

C and N stable isotope signatures reveal constraints to nutritional modes in orchids from the Mediterranean and Macaronesia

We compared the nutritional modes and habitats of orchids (e.g., autotrophic, partially or fully mycoheterotrophic) of the Mediterranean region and adjacent islands of Macaronesia. We hypothesized that ecological factors (e.g., relative light availability, surrounding vegetation) determine the nutritional modes of orchids and thus impose restrictions upon orchid distribution. Covering habitats from dark forests to open sites, orchid samples of 35 species from 14 genera were collected from 20 locations in the Mediterranean and Macaronesia to test for mycoheterotrophy. Mycorrhizal fungi were identified via molecular analyses, and stable isotope analyses were applied to test whether organic nutrients are gained from the fungal associates. Our results show that orchids with partial or full mycoheterotrophy among the investigated species are found exclusively in Neottieae thriving in light-limited forests. Neottioid orchids are missing in Macaronesia, possibly because mycoheterotrophy is constrained by the lack of suitable ectomycorrhizal fungi. Furthermore, most adult orchids of open habitats in the Mediterranean and Macaronesia show weak or no N gains from fungi and no C gain through mycoheterotrophy. Instead isotope signatures of some of these species indicate net plant-to-fungus C transfer.

Gene expression in mycorrhizal orchid protocorms suggests a friendly plant–fungus relationship

AbstractMain conclusionOrchid mycorrhiza has been often interpreted as an antagonistic relationship. Our data on mycorrhizal protocorms do not support this view as plant defence genes were not induced, whereas some nodulin-like genes were significantly up-regulated. Orchids fully depend on symbiotic interactions with specific soil fungi for seed germination and early development. Germinated seeds give rise to a protocorm, a heterotrophic organ that acquires nutrients, including organic carbon, from the mycorrhizal partner. It has long been debated if this interaction is mutualistic or antagonistic. To investigate the molecular bases of the orchid response to mycorrhizal invasion, we developed a symbiotic in vitro system between Serapias vomeracea, a Mediterranean green meadow orchid, and the rhizoctonia-like fungus Tulasnella calospora. 454 pyrosequencing was used to generate an inventory of plant and fungal genes expressed in mycorrhizal protocorms, and plant genes could be reliably identified with a customized bioinformatic pipeline. A small panel of plant genes was selected and expression was assessed by real-time quantitative PCR in mycorrhizal and non-mycorrhizal protocorm tissues. Among these genes were some markers of mutualistic (e.g. nodulins) as well as antagonistic (e.g. pathogenesis-related and wound/stress-induced) genes. None of the pathogenesis or wound/stress-related genes were significantly up-regulated in mycorrhizal tissues, suggesting that fungal colonization does not trigger strong plant defence responses. In addition, the highest expression fold change in mycorrhizal tissues was found for a nodulin-like gene similar to the plastocyanin domain-containing ENOD55. Another nodulin-like gene significantly more expressed in the symbiotic tissues of mycorrhizal protocorms was similar to a sugar transporter of the SWEET family. Two genes coding for mannose-binding lectins were significantly up-regulated in the presence of the mycorrhizal fungus, but their role in the symbiosis is unclear.

Pectin localization in the Mediterranean orchid Limodorum abortivum reveals modulation of the plant interface in response to different mycorrhizal fungi

In most mycorrhizal symbioses, phylogenetically distinct fungi colonize simultaneously the roots of individual host plants. A matter of debate is whether plants can distinguish among these fungal partners and differentiate their cellular responses. We have addressed this question in the orchid mycorrhizal symbiosis, where individual roots of the Mediterranean species Limodorum abortivum can be colonized by a dominant unculturable fungal symbiont belonging to the genus Russula and by more sporadic mycelia in the genus Ceratobasidium (form-genus Rhizoctonia). The phylogenetic position of the Ceratobasidium symbionts was further investigated in this work. Both Russula and Ceratobasidium symbionts form intracellular coils in the cortical roots of L. abortivum, but hyphae are very different in size and morphology, making the two fungi easily distinguishable. We have used John Innes Monoclonal 5, a widely used monoclonal antibody against pectin, to investigate the composition of the symbiotic plant interface around the intracellular coils formed by the two fungal partners. Immunolabelling experiments showed that pectin is exclusively found in the interface formed around the Ceratobasidium, and not around the Russula symbiont. These data indicate that the plant responses towards distinct mycorrhizal fungal partners can vary at a cellular level.

Hoya danumensis, a new species of Hoya (Apocynaceae, Asclepiadoideae) from Borneo

Summary The new species, Hoya danumensis, from Borneo is described and illustrated. It is proposed that its closest relationships may be found with Hoya campanulata with whom it shares the campanulate corolla.

Temporal variation in mycorrhizal diversity and carbon and nitrogen stable isotope abundance in the wintergreen meadow orchid Anacamptis morio.

Many adult orchids, especially photoautotrophic species, associate with a diverse range of mycorrhizal fungi, but little is known about the temporal changes that might occur in the diversity and functioning of orchid mycorrhiza during vegetative and reproductive plant growth. Temporal variations in the spectrum of mycorrhizal fungi and in stable isotope natural abundance were investigated in adult plants of Anacamptis morio, a wintergreen meadow orchid. Anacamptis morio associated with mycorrhizal fungi belonging to Tulasnella, Ceratobasidium and a clade of Pezizaceae (Ascomycetes). When a complete growing season was investigated, multivariate analyses indicated significant differences in the mycorrhizal fungal community. Among fungi identified from manually isolated pelotons, Tulasnella was more common in autumn and winter, the pezizacean clade was very frequent in spring, and Ceratobasidium was more frequent in summer. By contrast, relatively small variations were found in carbon (C) and nitrogen (N) stable isotope natural abundance, A. morio samples showing similar (15)N enrichment and (13)C depletion at the different sampling times. These observations suggest that, irrespective of differences in the seasonal environmental conditions, the plant phenological stages and the associated fungi, the isotopic content in mycorrhizal A. morio remains fairly constant over time.

Cryopreservation of orchid mycorrhizal fungi: A tool for the conservation of endangered species

The effectiveness of cryopreservation at --80 °C on orchid mycorrhizal fungi was assessed by testing the symbiotic ability of ten fungal isolates following cryo-storage for 10-24 months. The results obtained prove the efficacy of the method, thus providing a valuable tool for ex situ conservation.

The taxonomy of Hoya micrantha and Hoya revoluta (Apocynaceae, Asclepiadoideae)

Hoya revoluta, Hoya micrantha, and their synonyms are typified and revised. Hoya plicata, currently a synonym of H. micrantha, is reinstated and applied to a taxon from montane forests of Peninsular Malaysia; Hoya rundumensis is proposed to replace Hoya plicata subsp. rundumensis, a Bornean endemic; Hoya inconspicua is separated once again from Hoya revoluta; a new species, Hoya beccarii, is recognised and described.

Hoya corymbosa (Apocynaceae, Asclepiadoideae), A New Unusual Species from Sabah, Borneo, and Its Systematic Position Based on Phylogenetic Analysis

Abstract A new species, Hoya corymbosa, from Sabah (Borneo), is described and illustrated. It is a horizontally growing epiphytic shrub with diminutive flowers. Its minute staminal corona lobes lacking revolute outer margins and broad, flat caudicles of the pollinarium (resembling those of Dischidia) are quite peculiar within the genus, which is otherwise characterized by corona lobes with revolute margins and generally round to oval caudicles. Its position in Hoya has been confirmed by phylogenetic analysis based on nuclear ribosomal ITS region, the matK gene and psbA-trnH intergenic spacer.

Hoya devogelii (Apocynaceae Asclepiadoideae), a new species from kerangas heath forests in Sarawak, Borneo

Summary The new species Hoya devogelii Rodda & Simonsson with campanulate corollas from kerangas montane forest of Sarawak, Borneo is here described and illustrated. This species differs from any previously described species of Hoya in the very pubescent corolla and the staminal corona with a very developed process forming a ring around the base of the corona lobes. Morphological affinities between H. devogelii and other campanulate flowered species of Hoya from Borneo are discussed.