Enrico Ercole

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.

Molecular phylogeny, morphology, pigment chemistry and ecology in Hygrophoraceae (Agaricales)

Molecular phylogenies using 1–4 gene regions and information on ecology, morphology and pigment chemistry were used in a partial revision of the agaric family Hygro- phoraceae. The phylogenetically supported genera we recognize here in the Hygrophoraceae based on these and previous analyses are: Acantholichen, Ampulloclitocybe, Arrhenia, Cantharellula, Cantharocybe, Chromosera, Chrysomphalina, Cora, Corella, Cuphophyllus, Cyphellostereum, Dictyonema, Eonema, Gliophorus, Haasiella, Humidicutis, Hygroaster, Hygrocybe, Hygrophorus, Lichenomphalia, Neohygrocybe, Porpolomopsis and Pseudoarmillariella. A new genus that is sister to Chromosera is described as Gloioxanthomyces. Revisions were made at the ranks of subfamily, tribe, genus, subgenus, section and subsection. We present three new subfamilies, eight tribes (five new), eight subgenera (one new, one new combination and one stat. nov.), 26 sections (five new and three new combinations and two stat. nov.) and 14 subsections (two new, two stat. nov.). Species of Chromosera, Gliophorus, Humidicutis, and Neohygrocybe are often treated within the genus Hygrocybe; we therefore provide valid names in both classification systems. We used a minimalist approach in transferring genera and creating new names and combinations. Consequently, we retain in the Hygrophoraceae the basal cuphophylloid grade comprising the genera Cuphophyllus, Ampulloclitocybe and Cantharocybe, despite weak phylogenetic support. We include Aeruginospora and Semiomphalina in Hygrophoraceae based on morphology though molecular data are lacking. The lower hygrophoroid clade is basal to Hygrophoraceae s.s., comprising the genera Aphroditeola, Macrotyphula, Phyllotopsis, Pleurocybella, Sarcomyxa, Tricholomopsis and Typhula.

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.

Dothideomycetes and Leotiomycetes sterile mycelia isolated from the Italian seagrass Posidonia oceanica based on rDNA data.

Marine fungi represent a group of organisms extremely important from an ecological and biotechnological point of view, but often still neglected. In this work, an in-depth analysis on the systematic and the phylogenetic position of 21 sterile mycelia, isolated from Posidonia oceanica, was performed.The molecular (ITS and LSU sequences) analysis showed that several of them are putative new species belonging to three orders in the Ascomycota phylum: Pleosporales, Capnodiales and Helotiales. Phylogenetic analyses were performed using Bayesian Inference and Maximum Likelihood approaches.Seven sterile mycelia belong to the genera firstly reported from marine environments.The bioinformatic analysis allowed to identify five sterile mycelia at species level and nine at genus level. Some of the analyzed sterile mycelia could belong to new lineages of marine fungi.

A new taxon in the Infundibulicybe gibba complex (Basidiomycota, Agaricales, Tricholomataceae) from Sardinia (Italy).

A new species of Infundibulicybe (viz. I. mediterranea sp. nov.) is described from Sardinia based both on morphological and molecular ITS data. The species, a close ally of I. gibba, differs from the latter in the darker tinges of the basidiomata, the stipe, which is nearly concolorous with the pileus, and smaller basidiospores. Drawings of the main micro-morphological features as well as a color photograph of fresh basidiomata in situ are provided.

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.

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.

A preliminary ITS phylogeny of Melanoleuca (Agaricales), with special reference to European taxa

Ninety-one Melanoleuca collections were chosen to test the agreement of current and traditional infrageneric tripartite classifications of Melanoleuca (subgenera Acystis without cystidia, Urticocystis with urticiform cystidia, and Melanoleuca with macrocystidia) with molecular phylogenetic data (ITS sequences analysis) and to evaluate the systematic significance of relevant morphological characters. Melanoleuca is found to be monophyletic, and only two emended subgenera, Urticocystis and Melanoleuca, are supported. Subg. Urticocystis comprises all taxa with urticoid cystidia plus the macrocystidiate M. cognata complex. The artificial subg. Acystis is shown to be polyphyletic and no longer tenable. The entire genus comprises at least 10 clades with 13 subclades. Melanoleuca sublanipes sp. nov. and new combinations M. exscissa f. iris, M. exscissa f. sarcophylla, M. exscissa f. diverticulata, and M. paedida f. electropoda are introduced.

A new Neopaxillus species (Agaricomycetes) from the Dominican Republic and the status of Neopaxillus within the Agaricales

The new species Neopaxillus dominicanus is described on the basis of collections from the Dominican Republic. It is distinguished by having a basidiome with decurrent, distant, white lamellae with evident pink-lilac tinges, a non-depressed pileus at maturity and well developed catenulate cheilocystidia. A description, color photographs of fresh basidiomes and line drawings of relevant microscopic traits are provided. N. dominicanus is morphologically similar to Neopaxillus echinospermus, a type species of the genus. Based on comparative ITS-LSU rDNA gene sequence analyses, Neopaxillus, formerly placed in the Boletales, is considered within the Agaricales where it is sister to Crepidotus (Crepidotaceae), and N. dominicanus is supported as distinct from N. echinospermus. Finally, according to our morphological and molecular analyses, two collections of N. echinospermus from Mexico are referable to N. dominicanus.