Juan Pablo Suárez

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.

Mycorrhizal Associations in Ferns from Southern Ecuador

Abstract We conducted a survey on the mycorrhizal status of neotropical ferns, focusing on previously neglected taxa. These include the filmy ferns (Hymenophyllaceae), grammitid ferns (Polypodiaceae), and the genus Elaphoglossum (Dryopteridaceae). Samples were collected at different sites in southern Ecuador, Prov. Loja, Morona-Santiago, and Zamora-Chinchipe. Among the 85 investigated species (101 samples, 10 families), 19 were associated with arbuscular mycorrhizal fungi (AMF) and 36 were infected by dark septate endophytes (DSE), which are identified as ascomycetes and here considered as a kind of mycorrhiza similar to the ericoid type. The roots of 30 species (including all non-grammitid Polypodiaceae and half of the Elaphoglossum species) were free of evident fungal infection. AMF were frequent in terrestrial species (29.10% of species, or 48.49% of infected terrestrial samples). DSE prevailed in epiphytic species (58.62% of species, or 96.15% of infected epiphytic samples) and were also common in terrestrial samples of predominantly epiphytic species.

A clustering optimization strategy to estimate species richness of Sebacinales in the tropical Andes based on molecular sequences from distinct DNA regions

Fungi are believed to be diverse in the tropics, but because many groups are only known from their DNA sequences this hampers comparative diversity studies. We investigated mycorrhizal Sebacinales (Basidiomycota) of 67 individuals of Ericaceae and Orchidaceae in a tropical mountain ecosystem in Southern Ecuador to provide a first estimate of whether these fungi are particularly diverse in the Northern Andes. We partially sequenced the internal transcribed spacer (ITS) and large subunit (LSU) regions of the nuclear ribosomal DNA and analyzed them together with all Sebacinales sequences available from GenBank. The clustering optimization technique was used to determine clustering parameters that maximize the comparability between molecular operational taxonomic units (MOTUs) obtained from the distinct loci. Sampling effort and species richness were estimated with rarefaction-accumulation curves and non-parametric estimation using Chao2 and compared between Southern Ecuador and France. Clustering optimization indicated that a 1% LSU distance threshold corresponds to the commonly used 3% dissimilarity threshold for ITS, and that a clustering algorithm close to single-linkage clustering is optimal. The resulting clusters show that about 8–9% of observed Sebacinales MOTUs occur in the study area and that most of these MOTUs are endemic (74%). The widespread MOTUs from Southern Ecuador were also found in Panama, North America and Europe. The estimation of species richness revealed unsaturated sampling of Sebacinales in general and also in our study area. Our results suggest a high diversity of Sebacinales associated with Ericaceae and Orchidaceae at the study site in Southern Ecuador, but no hotspot of Sebacinales in comparison with other areas.

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.

Reforestation sites show similar and nested AMF communities to an adjacent pristine forest in a tropical mountain area of South Ecuador.

Arbuscular mycorrhizae are important for growth and survival of tropical trees. We studied the community of arbuscular mycorrhizal fungi in a tropical mountain rain forest and in neighbouring reforestation plots in the area of Reserva Biológica San Francisco (South Ecuador). The arbuscular mycorrhizal fungi were analysed with molecular methods sequencing part of the 18 S rDNA. The sequences were classified as Operational Taxonomic Units (OTUs). We found high fungal species richness with OTUs belonging to Glomerales, Diversisporales and Archaeosporales. Despite intensive sampling, the rarefaction curves are still unsaturated for the pristine forest and the reforestation plots. The communities consisted of few frequent and many rare species. No specific interactions are recognizable. The plant individuals are associated with one to ten arbuscular mycorrhizal fungi and mostly with one to four. The fungal compositions associated with single plant individuals show a great variability and variety within one plant species. Planted and naturally occurring plants show high similarities in their fungal communities. Pristine forest and reforestation plots showed similar richness, similar diversity and a significantly nested structure of plant-AMF community. The results indicate that small-scale fragmentation presently found in this area has not destroyed the natural AMF community, at least yet. Thus, the regeneration potential of natural forest vegetation at the tested sites is not inhibited by a lack of appropriate mycobionts.

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.

Identification of mVOCs from Andean Rhizobacteria and Field Evaluation of Bacterial and Mycorrhizal Inoculants on Growth of Potato in its Center of Origin

Food security (a pressing issue for all nations) faces a threat due to population growth, land availability for growing crops, a changing climate (leading to increases in both abiotic and biotic stresses), heightened consumer awareness of the risks related to the use of agrichemicals, and also the reliance on depleting fossil fuel reserves for their production. Legislative changes in Europe mean that fewer agrichemicals will be available in the future for the control of crop pests and pathogens. The need for the implementation of a more sustainable agricultural system globally, incorporating an integrated approach to disease management, has never been more urgent. To that end, the Valorizing Andean Microbial Diversity (VALORAM) project (http://valoram.ucc.ie), funded under FP7, examined the role of microbial communities in crop production and protection to improve the sustainability, food security, environmental protection, and productivity for rural Andean farmers. During this work, microbial volatile organic compounds (mVOCs) of 27 rhizobacterial isolates were identified using gas chromatography/mass spectrometry (GC/MS), and their antifungal activity against Rhizoctonia solani was determined in vitro and compared to the activity of a selection of pure volatile compounds. Five of these isolates, Pseudomonas palleroniana R43631, Bacillus sp. R47065, R47131, Paenibacillus sp. B3a R49541, and Bacillus simplex M3-4 R49538 trialled in the field in their respective countries of origin, i.e., Bolivia, Peru, and Ecuador, showed significant increase in the yield of potato. The strategy followed in the VALORAM project may offer a template for the future isolation and determination of putative biocontrol and plant growth-promoting agents, useful as part of a low-input integrated pest management system.

Orchids keep the ascomycetes outside: a highly diverse group of ascomycetes colonizing the velamen of epiphytic orchids from a tropical mountain rainforest in Southern Ecuador

Orchid mycorrhizal detection, based only on fungal isolation from roots, is biased due to difficulties in isolating the respective fungi. Previous investigations have shown that mostly ascomycetes, housed in the sheltered compartment of the velamen covering the roots of epiphytic orchids, are isolated on agar plates. Roots of 83 individual epiphytic orchid of Stelis hallii, S. superbiens, S. concinna, Stelis sp. and Pleurothallis lilijae were sampled in the Reserva Biológica San Francisco, Southern Ecuador. The velamen was partly removed and root tissue macerated and transferred to Petri dishes containing corn meal agar, malt extract agar and Melin Norkrans modified medium. DNA was extracted from the cultures and the 5.8S-ITS region and partial nrLSU sequenced. Phylogenetic analysis revealed members of 12 orders of ascomycetes associated with the roots of these orchids, a much higher diversity than previously known. Twelve isolates were related to the Helotiales. Our results have uncovered some of the fungal diversity within the velamen of epiphytic orchids; fungi invading, most likely, from tree bark or humus accumulation close to the roots.

Do mycorrhizal fungi drive speciation in Teagueia (Orchidaceae) in the upper Pastaza watershed of Ecuador

The orchid genus Teagueia Luer (Orchidaceae, subtribe Pleurothallidinae) presents an extraordinary example of recent local evolutionary radiation. In principle, mutualisms might affect the origin of plant species via an effect on speciation. As orchids depend on mycorrhizal fungi for seed germination and early plantlet development we tested whether certain mycorrhizal fungi are acting as drivers of this radiation in Teagueia species. Sampling was carried out near Baños in east Andean Ecuador. Roots were collected from a total of 11 flowering individuals of eight morphospecies (referred to as Teagueia spp). The whole ITS1-5.8S- ITS2 nrDNA region and part of the 28S nrDNA were amplified, cloned and sequenced. Molecular phylogeny of the obtained sequences revealed four phylogenetic species of Tulasnellaceae and one of Atractiellales (Pucciniomycotina, Basidiomycota) associated with Teagueia spp. Tulasnelloid fungi were detected in all samples. Up to three different phylogenetic species of mycobionts were found associated with one Teagueia species. We found that co-occurring Teagueia species share mycobionts. All detected mycobionts had wide geographical distribution. Based on the available evidence we conclude that the extraordinary local radiation of Teagueia is most likely driven by other factors than by mycorrhizal fungi, but that mycorrhiza may be a key factor for the coexistence of so many closely related orchid species.

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.