Darío Cruz

Atractiellomycetes belonging to the ‘rust’ lineage (Pucciniomycotina) form mycorrhizae with terrestrial and epiphytic neotropical orchids

Distinctive groups of fungi are involved in the diverse mycorrhizal associations of land plants. All previously known mycorrhiza-forming Basidiomycota associated with trees, ericads, liverworts or orchids are hosted in Agaricomycetes, Agaricomycotina. Here we demonstrate for the first time that Atractiellomycetes, members of the ‘rust’ lineage (Pucciniomycotina), are mycobionts of orchids. The mycobionts of 103 terrestrial and epiphytic orchid individuals, sampled in the tropical mountain rainforest of Southern Ecuador, were identified by sequencing the whole ITS1-5.8S-ITS2 region and part of 28S rDNA. Mycorrhizae of 13 orchid individuals were investigated by transmission electron microscopy. Simple septal pores and symplechosomes in the hyphal coils of mycorrhizae from four orchid individuals indicated members of Atractiellomycetes. Molecular phylogeny of sequences from mycobionts of 32 orchid individuals out of 103 samples confirmed Atractiellomycetes and the placement in Pucciniomycotina, previously known to comprise only parasitic and saprophytic fungi. Thus, our finding reveals these fungi, frequently associated to neotropical orchids, as the most basal living basidiomycetes involved in mycorrhizal associations of land plants.

Defining species in Tulasnella by correlating morphology and nrDNA ITS-5.8S sequence data of basidiomata from a tropical Andean forest

The genus Tulasnella comprises important orchid mycobionts. Molecular phylogenetic studies on nrITS-5.8S sequences of Tulasnella species previously isolated from mycorrhizas of epiphytic orchids from a tropical Andean forest showed genomic variability among clones which was difficult to interpret as intra- or interspecific variations or to correlate with described Tulasnella species. To improve this situation, we collected basidiomata of Tulasnella in an Andean forest, studied part of the sequences of fungal ribosomal genes and correlated molecular data with the morphology of the specimens. Within five basidiomata displaying slight morphological variability, we found inter-specimen nrITS1-5.8S-ITS2 variability corresponding to proportional differences of less than 1% except for one clone with 5.1% divergence. Results indicate that the slightly variable basidiomata should be considered as one species, which is morphologically tentatively assigned to the Tulasnella pruinosa complex. However, comparison of nrITS1-5.8S-ITS2 sequences, including sequences of T. pruinosa from other origins, indicate that Tulasnella sp. is only distantly related to the T. pruinosa specimens included in the analyses. Sequences of all morphologically similar and taxonomically well-identified species are required to decide whether the basidiomata analyzed in the present study represent a new species. The new sequences are rather similar to sequences obtained previously from mycorrhizae of epiphytic orchids of the same area indicating mycorrhizal potential of this fungus.

Mycorrhiza Networks Promote Biodiversity and Stabilize the Tropical Mountain Rain Forest Ecosystem: Perspectives for Understanding Complex Communities

To better understand the mechanisms behind maintenance of the extraordinary plant and fungal diversity in tropical mountain forests we applied, for the first time, network theory to investigate the mycobiont–plant communities. We addressed three different mycorrhizal classes, arbuscular mycorrhizae of tropical trees, mycorrhizae of terrestrial and epiphytic Orchidaceae and cavendishioid mycorrhizae among Ericaceae and Sebacinales. We found significant nestedness (NODF) for arbuscular and orchid mycorrhizal networks. In accordance to previous simulations and verifications of species-rich, mutualistic plant–animal networks, we conclude that preferential attachment of new members to already existing links integrates and maintains rare species and stabilizes our species rich assemblages.

Cryptic species revealed by molecular phylogenetic analysis of sequences obtained from basidiomata of Tulasnella

Delimitation of species and the search for a proper threshold for defining phylogenetic species in fungi are under discussion. In this study, morphological and molecular data are correlated to delimit species of Tulasnella, the most important mycobionts of Orchidaceae, which suffer from poor taxonomy. Resupinate basidiomata of Tulasnella species were collected in Ecuador and Germany, and 11 specimens (seven from Ecuador, four from Germany) were assigned to traditional species concepts by use of morphological keys. The specimens were compared by micro-anatomical examination with 75 specimens of Tulasnella borrowed from fungaria to obtain better insights on variation of characters. Sequences of the ITS region (127) were obtained after cloning from the fresh basidiomata and from pure cultures. Proportional variability of ITS sequences was analyzed within and among the cultures and the specimens designated to different morphospecies. Results suggested an intragenomic variation of less than 2%, an intraspecific variation of up to 4% and an interspecific divergence of more than 9% in Tulasnella. Cryptic species in Tulasnella, mostly from Ecuador, were revealed by phylogenetic analyses with 4% intraspecific divergence as a minimum threshold for delimiting species. Conventional diagnostic morphological characters appeared insufficient for species characterization. Arguments are presented for molecular delimitation of the established species Tulasnella albida, T. asymmetrica, T. eichleriana, T. cf. pinicola, T. tomaculum and T. violea.

Mycorrhizal preferences and fine spatial structure of the epiphytic orchid Epidendrum rhopalostele

PREMISE OF THE STUDY The presence of compatible fungi is necessary for epiphytic orchid recruitment. Thus, identifying associated mycorrhizal fungi at the population level is essential for orchid conservation. Recruitment patterns may also be conditioned by factors such as seed dispersal range and specific environmental characteristics. METHODS In a forest plot, all trees with a diameter at breast height >1 cm and all individuals of the epiphytic orchid Epidendrum rhopalostele were identified and mapped. Additionally, one flowering individual of E. rhopalostele per each host tree was randomly selected for root sampling and DNA extraction. KEY RESULTS A total of 239 E. rhopalostele individuals were located in 25 of the 714 potential host trees. Light microscopy of sampled roots showed mycorrhizal fungi in 22 of the 25 sampled orchids. Phylogenetic analysis of ITS1-5.8S-ITS2 sequences yielded two Tulasnella clades. In four cases, plants were found to be associated with both clades. The difference between univariate and bivariate K functions was consistent with the random labeling null model at all spatial scales, indicating that trees hosting clades A and B of Tulasnella are not spatially segregated. The analysis of the inhomogenous K function showed that host trees are not clustered, suggesting no limitations to population-scale dispersal. χ(2) analysis of contingency tables showed that E. rhopalostele is more frequent on dead trees than expected. CONCLUSIONS EPIDENDRUM RHOPALOSTELE establishes mycorrhizal associations with at least two different Tulasnella species. The analysis of the distribution patterns of this orchid suggests a microsite preference for dead trees and no seed dispersal limitation.

Distinct but Closely Related Sebacinales form Mycorrhizae with Coexisting Ericaceae and Orchidaceae in a Neotropical Mountain Area

Sebacinales form mycorrhizae with a variety of plant families including Ericaceae and Orchidaceae. Structurally, these mutual associations are quite distinct, with specific structures largely depending on the individual plant family. Many plants that are associated with Sebacinales occur in the same habitats and it has been questioned whether they share a common pool of mycobionts or are associated with different Sebacinales guilds.

Biogeography and Ecology of Tulasnellaceae

Species of Tulasnellaceae share the unique feature of tulasnelloid basidia, characterised by swollen sterigmata. All species have a resupinate inconspicuous or lacking basidiomata. Only three genera are assigned to the family, two of them being monospecific. Here, we treat the species of the genus Tulasnella phylogenetically, ecologically, and biogeographically and review the literature accordingly. Most geographic data are derived from floristic and molecular studies of tulasnelloid mycobionts in mycorrhizae. Tulasnella species have a world-wide distribution. They appear to be associated with orchids on a global scale, and may occur in many forest ecosystems as lignicolous fungi.

Antibacterial and cytotoxic activity from the extract and fractions of a marine derived bacterium from the Streptomyces genus

Abstract Context: Marine natural products are a rich source of potent, selective, and structurally novel compounds. Marine bacteria are considered the most promising source of biologically active compounds which can be applied to treat a wide range of diseases. Objective: The current study was designed to establish the bases for a future marine exploration in the Ecuadorian coast based on the molecular identification of a marine bacterium and its potential use as an antibacterial or cytotoxic compounds source. Materials and methods: Isolation and characterization of the marine bacterium were carried out through microbiological methods from desiccated sediment. Molecular identification was made by means of 16S rDNA analysis. MIC was measured by the microdilution broth method against six pathogenic bacteria: two Gram positive and four Gram negative strains. Cytotoxicity was evaluated by Crystal violet assay against breast adenocarcinoma (MCF7) and ductal carcinoma (T47D and ZR-75-30). Results: Our present study has shown that EtOAc extract and fraction A1 obtained from marine Streptomyces sp. revealed the maximal antibacterial and cytotoxic activity. Enterococcus faecalis was found to be more sensitive strain (MIC 0.78 μg/ml) than the other five bacteria tested. ZR-75-30 and T47D cell lines were found to be more sensitive (IC50 value, 31.88 ± 0.05 and 68.35 ± 0.12 μg/ml) than adenocarcinoma MCF7 (IC50 value was 83.65 ± 0.06 μg/ml). Discussion and conclusion: The results obtained herein indicate that EtOAc extract of Streptomyces sp. has shown a strong antibacterial activity as well as moderate cytotoxic activity which make it a good candidate for metabolite isolation.