Fine-scale spatial distribution of orchid mycorrhizal fungi in the soil of host-rich grasslands.

Mycorrhizal fungi are essential for the survival of orchid seedlings under natural conditions. The distribution of these fungi in soil can constrain the establishment and resulting spatial arrangement of orchids at the local scale, but the actual extent of occurrence and spatial patterns of orchid mycorrhizal (OrM) fungi in soil remain largely unknown. We addressed the fine-scale spatial distribution of OrM fungi in two orchid-rich Mediterranean grasslands by means of high-throughput sequencing of fungal ITS2 amplicons, obtained from soil samples collected either directly beneath or at a distance from adult Anacamptis morio and Ophrys sphegodes plants. Like ectomycorrhizal and arbuscular mycobionts, OrM fungi (tulasnelloid, ceratobasidioid, sebacinoid and pezizoid fungi) exhibited significant horizontal spatial autocorrelation in soil. However, OrM fungal read numbers did not correlate with distance from adult orchid plants, and several of these fungi were extremely sporadic or undetected even in the soil samples containing the orchid roots. Orchid mycorrhizal 'rhizoctonias' are commonly regarded as unspecialized saprotrophs. The sporadic occurrence of mycobionts of grassland orchids in host-rich stands questions the view of these mycorrhizal fungi as capable of sustained growth in soil.

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.

Cupreoboletus (Boletaceae, Boletineae), a new monotypic genus segregated from Boletus sect. Luridi to reassign the Mediterranean species B. poikilochromus.

Cupreoboletus is erected as a new monospecific genus of Boletaceae to accommodate the thermophilic southern European species Boletus poikilochromus, characterized by discoloration toward copper-red tints overall, hymenophore forming tiny crystals on drying, a pervasive and long lasting sweet odor and presence of pseudocystidia. Macro- and microscopic descriptions of the species based on re-examination of the type material and recent Italian collections including additional topotypical samples are provided and accompanied by photos and line drawings of the main anatomical structures. In addition, an epitype specimen is selected. Phylogenetic relationships were inferred from multigene molecular analysis based on partial sequences of the nuc rDNA 28S D1/D2 (28S) and internal transcribed spacer (ITS) regions and genes for ribosomal RNA polymerase II subunits 1 (rpb1) and 2 (rpb2) and translation-elongation factor 1-α (tef1α). Ecological context, geographical range and delimitation from closely allied taxa also were elucidated. B. martaluciae is treated as a synonym of C. poikilochromus according to the morphological and molecular comparative study.

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

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.

Alessioporus and Pulchroboletus (Boletaceae, Boletineae), two novel genera for Xerocomus ichnusanus and X. roseoalbidus from the European Mediterranean basin: molecular and morphological evidence.

Alessioporus and Pulchroboletus are proposed as new monotypic genera to accommodate the thermo-xerophilic European species Xerocomus ichnusanus and X. roseoalbidus respectively. The present research focused on both morphological features and multigene molecular phylogeny (nrITS, nrLSU, tef-1α datasets) to elucidate the taxonomic status of these two rare Mediterranean boletes and delineate a natural classification within the family Boletaceae. Macro- and microscopic descriptions of the two species based on inclusive taxon sampling are provided and supported by line drawings of the main anatomical features. Phylogenetic relationships, ecology, geographical distribution and delimitation from the most closely allied taxa also are highlighted. In addition, epitype specimens are selected for both species.

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.

The phylogenetic position of Haasiella (Basidiomycota, Agaricomycetes) and the relationships between H. venustissima and H. splendidissima.

Based on a combined ITS-LSU rDNA analysis, the omphalinoid genus Haasiella is shown to be part of the Hygrophoroid clade and sister to Hygrophorus. H. venustissima and H. splendidissima are conspecific; H. splendidissima is the tetrasporic/clamped and heterothallic form of H. venustissima.

A new Cortinarius of section Calochroi (Basidiomycota, Agaricomycetes) from Mediterranean Quercus woodlands (Italy).

A new species of Cortinarius, C. flavoaurantians sp. nov., is described from Italian Quercus woods based on both morphological and ITS rDNA data. This taxon is characterized by a yellowish pileus and cortina, a white universal veil and a pileipellis that reacts yellow-orange with KOH. Illustrations of the key micromorphological features and fresh basidiomata in situ are provided. Closely related species are discussed.

Alpine constructed wetlands: A metagenomic analysis reveals microbial complementary structure.

Constructed wetlands (CWs) are used to water treatment worldwide, however their application at high-altitude is poorly studied. In order to survive mountain winters, CWs rely on native flora and associated microbial communities. However, the choice of plant-microbes pairs more suitable for water treatment is challenging in alpine environments. Using a metagenomic approach, we investigated the composition of prokaryotes and fungal communities, through extensive sampling inside a constructed wetland in the SW-Alps. Best performing plant species were searched among those hosting the most diverse and resilient microbial communities and to this goal, we analysed them in the natural environment also. Our results showed that microbial communities were less diverse in the CW than at natural conditions, and they differed from plant to plant, revealing a clear variation in taxonomic composition between forbs and gramineous plants. Carex rostrata, Deschampsia caespitosa and Rumex alpinus hosted bacteria very active in N-cycles. Moreover, fungal and prokaryotic communities associated to R. alpinus (Polygonaceae) turned to be the richest and stable among the studied species. In our opinion, this species should be prioritized in CWs at high elevations, also in consideration of its low maintenance requirements.

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.

A new annulate Pluteus variety from Italy.

Pluteus aurantiorugosus, var. aurantiovelatus var. nov., is described from Italy based on both morphological and ITS rDNA data. This taxon is characterized by an ornamented stipe provided with reddish orange remnants of a partial veil. The occurrence of the partial veil in Pluteus also is discussed.

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.

Taxonomic Re-Examination of Nine Rosellinia Types (Ascomycota, Xylariales) Stored in the Saccardo Mycological Collection.

In a recent monograph on the genus Rosellinia, type specimens worldwide were revised and re-classified using a morphological approach. Among them, some came from Pier Andrea Saccardo's fungarium stored in the Herbarium of the Padova Botanical Garden. In this work, we taxonomically re-examine via a morphological and molecular approach nine different Roselliniasensu Saccardo types. ITS1 and/or ITS2 sequences were successfully obtained applying Illumina MiSeq technology and phylogenetic analyses were carried out in order to elucidate their current taxonomic position. Only the ITS1 sequence was recovered for Rosellinia areolata, while for R. geophila, only the ITS2 sequence was recovered. We proposed here new combinations for Rosellinia chordicola, R. geophila and R. horridula, while for R. ambigua, R. areolata, R. australis, R. romana and R. somala, we did not suggest taxonomic changes compared to the current ones. The name Rosellinia subsimilis Sacc. is invalid, as it is a later homonym of R. subsimilis P. Karst. & Starbäck. Therefore, we introduced Coniochaeta dakotensis as a nomen novum for R. subsimilis Sacc. This is the first time that these types have been subjected to a molecular study. Our results demonstrate that old types are an important source of DNA sequence data for taxonomic re-examinations.

Plasticity, exudation and microbiome-association of the root system of Pellitory-of-the-wall plants grown in environments impaired in iron availability.

The investigation of the adaptive strategies of wild plant species to extreme environments is a challenging issue, which favors the identification of new traits for plant resilience. We investigated different traits which characterize the root-soil interaction of Parietaria judaica, a wild plant species commonly known as "Pellitory-of-the-wall". P. judaica adopts the acidification-reduction strategy (Strategy I) for iron (Fe) acquisition from soil, and it can complete its life cycle in highly calcareous environments without any symptoms of chlorosis. In a field-to-lab approach, the microbiome associated with P. judaica roots was analyzed in spontaneous plants harvested from an urban environment consisting in an extremely calcareous habitat. Also, the phenolics and carboxylates content and root plasticity and exudation were analyzed in P. judaica plants grown under three different controlled conditions mimicking the effect of calcareous environments on Fe availability: results show that P. judaica differentially modulates root plasticity under different Fe availability-impaired conditions, and that it induces, to a high extent, the exudation of caffeoylquinic acid derivatives under calcareous conditions, positively impacting Fe solubility.

Outstanding Pinkish Brown-Spored Neotropical Boletes: Austroboletus subflavidus and Fistulinella gloeocarpa (Boletaceae, Boletales) from the Dominican Republic.

The occurrence of Austroboletus subflavidus and Fistulinella gloeocarpa is documented from the Dominican Republic. The latter species is reported for the first time outside its original locality in Martinique, extending the geographic range for this uncommon pinkish-spored bolete. A detailed morphological description is provided for each species and accompanied by color pictures of fresh basidiomes in habitat and line drawings of the main anatomical features. Both species represent independent lineages within their respective genera based on phylogenetic inference. In addition, A. subflavidus clusters in a sister lineage to the core Austroboletus clade (Austroboletus clade I) here named as Austroboletus clade II. In order to confirm the accuracy of species identification, their identity and relationships were subjected to multilocus phylogenetic analyses of three gene markers (ITS, nrLSU, RPB2) including genetic material already available in public databases. Austroboletus subflavidus is a widely distributed species in North and Central America, whereas F. gloeocarpa is apparently highly localized and seems to appear sparingly in the Dominican Republic, Martinque, and southern Florida. Comparisons with morphologically similar and molecularly inferred allied species are also presented and discussed.

Looking for Lepiotapsalion Huijser & Vellinga (Agaricales, Agaricaceae).

Lepiotapsalion is fully described based on a recent collection from Sardinia (Italy) and the holotype. NrITS- and nrLSU-based phylogeny demonstrates that sequences deposited in GenBank as "L.psalion" and generated from two Dutch and one Chinese collections are not conspecific with the holotype and represent two distinct, undescribed species. These species are here proposed as Lepiotarecondita sp. nov. and Lepiotasinorecondita ad int.

A new species of Rhodocybe sect. Rufobrunnea (Entolomataceae, Agaricales) from Italy.

Rhodocybe fumanellii is described from Italy as a new species based both on morphological and molecular nrITS/nrLSU data. It belongs in sect. Rufobrunnea and is characterised by massive tricholomatoid basidiomata with reddish-brown tinges, adnate and crowded lamellae, an enlarged stipe base with long rhizomorphs, long sinuose slender cheilocystidia, ellipsoid basidiospores and the presence of caulocystidia. Drawings of the main micromorphological features as well as a colour photograph of fresh basidiomata in situ are provided and its morphological relationships with allied species are discussed.

Diversity of polypores in the Dominican Republic: Pseudowrightoporia dominicana sp. nov. (Hericiaceae, Russulales).

The new species Pseudowrightoporia dominicana is described from the Dominican Republic based on morphological and molecular data (nrITS and nrLSU sequence analyses). It is mainly characterised by pileate basidiomata with a bright pinkish context and a di-trimitic hyphal system. Phylogenetically, it is sister to the African species P. gillesii and to the Asiatic P. japonica.

The abundance and diversity of arbuscular mycorrhizal fungi are linked to the soil chemistry of screes and to slope in the Alpic paleo-endemic Berardia subacaulis.

Berardia subacaulis Vill. is a monospecific genus that is endemic to the South-western Alps, where it grows on alpine screes, which are extreme habitats characterized by soil disturbance and limiting growth conditions. Root colonization by arbuscular mycorrhizal fungi (AMF) is presumably of great importance in these environments, because of its positive effect on plant nutrition and stress tolerance, as well as on structuring the soil. However, there is currently a lack of information on this topic. In this paper, we tested which soil characteristics and biotic factors could contribute to determining the abundance and community composition of AMF in the roots of B. subacaulis, which had previously been found to be mycorrhizal. For such a reason, the influence of soil properties and environmental factors on AMF abundance and community composition in the roots of B. subacaulis, sampled on three different scree slopes, were analysed through microscopic and molecular analysis. The results have shown that the AMF community of Berardia roots was dominated by Glomeraceae, and included a core of AMF taxa, common to all three scree slopes. The vegetation coverage and dark septate endophytes were not related to the AMF colonization percentage and plant community did not influence the root AMF composition. The abundance of AMF in the roots was related to some chemical (available extractable calcium and potassium) and physical (cation exchange capacity, electrical conductivity and field capacity) properties of the soil, thus suggesting an effect of AMF on improving the soil quality. The non-metric multidimensional scaling (NMDS) ordination of the AMF community composition showed that the diversity of AMF in the various sites was influenced not only by the soil quality, but also by the slope. Therefore, the slope-induced physical disturbance of alpine screes may contribute to the selection of disturbance-tolerant AMF taxa, which in turn may lead to different plant-fungus assemblages.