Diversity of Agaricomycetes in southern South America and their bioactive natural products.

Fungi have a unique metabolic plasticity allowing them to produce a wide range of natural products. Since the discovery of penicillin, an antibiotic of fungal origin, substantial efforts have been devoted globally to search for fungal-derived natural bioactive products. Andean region forests represent one of the few undisturbed ecosystems in the world with little human intervention. While these forests display a rich biological diversity, mycological and chemical studies in these environments have been scarce. This review aims to summarise all the efforts regarding the chemical or bioactivity analyses of Agaricomycetes (Basidiomycota) from southern South America environments. Overall, herein we report a total of 147 fungal species, 21 of them showing chemical characterisation and/or biological activity. In terms of chemical cores, furans, chlorinated phenol derivatives, polyenes, lactones, terpenes and himanimides have been reported. These natural products displayed a range of biological activities including antioxidant, antimicrobial, antifungal, neuroprotective and osteoclast-forming suppressing effects.

Biogeographic history of a large clade of ectomycorrhizal fungi, the Russulaceae, in the Neotropics and adjacent regions.

The biogeography of neotropical fungi remains poorly understood. Here, we reconstruct the origins and diversification of neotropical lineages in one of the largest clades of ectomycorrhizal fungi in the globally widespread family Russulaceae. We inferred a supertree of 3285 operational taxonomic units, representing worldwide internal transcribed spacer sequences. We reconstructed biogeographic history and diversification and identified lineages in the Neotropics and adjacent Patagonia. The ectomycorrhizal Russulaceae have a tropical African origin. The oldest lineages in tropical South America, most with African sister groups, date to the mid-Eocene, possibly coinciding with a boreotropical migration corridor. There were several transatlantic dispersal events from Africa more recently. Andean and Central American lineages mostly have north-temperate origins and are associated with North Andean uplift and the general north-south biotic interchange across the Panama isthmus, respectively. Patagonian lineages have Australasian affinities. Diversification rates in tropical South America and other tropical areas are lower than in temperate areas. Neotropical Russulaceae have multiple biogeographic origins since the mid-Eocene involving dispersal and co-migration. Discontinuous distributions of host plants may explain low diversification rates of tropical lowland ectomycorrhizal fungi. Deeply diverging neotropical fungal lineages need to be better documented.

Exploring interactions between Beauveria and Metarhizium strains through co-inoculation and responses of perennial ryegrass in a one-year trial.

Perennial ryegrass (Lolium perenne L.) possesses a high level of nutritional quality and is widely used as a forage species to establish permanent pastures in southern Chile. However, the productivity of most such pastures is limited by various environmental agents, such as insect pests and drought. In this context, our work stresses the need for elucidating the ability of fungal endophytes to establish interactions with plants, and to understand how these processes contribute to plant performance and fitness. Therefore, we evaluated the colonization and impact of two native strains of the endophytic insect-pathogenic fungus (EIPF) group isolated from permanent ryegrass pastures in southern Chile. Roots and seeds of ryegrass and scarabaeid larvae were collected from nine different ryegrass pastures in the Los Ríos region of southern Chile to specifically isolate EIPFs belonging to the genera Beauveria and Metarhizium. Fungal isolations were made on 2% water agar with antibiotics, and strains were identified by analyzing the entire internal transcribed spacer (ITS) 1-5.8S-ITS2 ribosomal DNA region. Four strains of Beauveria and 33 strains of Metarhizium were isolated only in scarabaeid larvae from ryegrass pastures across four sites. Experimental mini-pastures that were either not inoculated (control) or co-inoculated with conidia of the strains Beauveria vermiconia NRRL B-67993 (P55_1) and Metarhizium aff. lepidiotae NRRL B-67994 (M25_2) under two soil humidity levels were used. Ryegrass plants were randomly collected from the mini-pastures to characterize EIPF colonization in the roots by real-time PCR and fluorescence microscopy. Aboveground biomass was measured to analyze the putative impact of colonization on the mini-pastures' aboveground phenotypic traits with R software using a linear mixed-effects model and the ANOVA statistical test. Seasonal variation in the relative abundance of EIPFs was observed, which was similar between both strains from autumn to spring, but different in summer. In summer, the relative abundance of both EIPFs decreased under normal moisture conditions, but it did not differ significantly under water stress. The aboveground biomass of ryegrass also increased from autumn to spring and decreased in summer in both the inoculated and control mini-pastures. Although differences were observed between moisture levels, they were not significant between the control and inoculated mini-pastures, except in July (fresh weight and leaf area) and October (dry weight). Our findings indicate that native strains of B. vermiconia NRRL B-67993 (P55_1) and M. aff. lepidiotae NRRL B-67994 (M25_2) colonize and co-exist in the roots of ryegrass, and these had little or no effect on the mini-pastures' aboveground biomass; however, they could have other functions, such as protection against root herbivory by insect pests.

Phylogenetic relationships in the genus Podoserpula and description of P. aliweni, a new species from Chile.

Phylogenetic analyses based on a three-locus nuclear data set (ITS1-5.8S-ITS2, the 5' end of the 28S, and the largest subunit of RNA polymerase I) supported the pagoda fungus (Podoserpula, Amylocorticiales) as a monophyletic group most closely related to species of Anomoporia, which is nonmonophyletic, and Amyloathelia. Phylogenetic relationships inferred from internal transcribed spacer (ITS) sequences of specimens sampled in Australia, Chile, China, Madagascar, and New Zealand divided Podoserpula into two major lineages: Clade A containing Australian and New Zealand collections designated P. pusio and the Chinese species P. ailaoshanensis, which have basidiospores with no reaction to Melzer's reagent, and Clade B, which includes a species described from Chile, P. aliweni, and specimens originating from Australia, Chile, Madagascar, and New Zealand with dextrinoid basidiospores. Podoserpula aliweni forms a unique branch in the phylogenetic tree and differs from its most closely related taxon by 1.8-2.1% in the ITS region. The new species exhibits a tree-like habit with a white to concolorous stipe-like base with the hymenophore's main subcylindrical axis bearing up to 18 superimposed pilei, slightly enrolled white margins shading from yellowish white to orange-yellow toward the center, and ellipsoid to broadly ellipsoid basidiospores measuring 4.0-4.5 × 3.5 µm. Ecologically, P. aliweni occurs during the rainy season, often gregariously on dried branches or wet soil under Nothofagus dombeyi or N. obliqua and has a distribution range of more than 600 km in southern Chile. This study extends the known distributional range and increases our knowledge on the phylogenetic diversity and taxonomy in Podoserpula.

Belowground fungal community diversity, composition and ecological functionality associated with winter wheat in conventional and organic agricultural systems.

Understanding the impacts of agricultural practices on belowground fungal communities is crucial in order to preserve biological diversity in agricultural soils and enhance their role in agroecosystem functioning. Although fungal communities are widely distributed, relatively few studies have correlated agricultural production practices. We investigated the diversity, composition and ecological functionality of fungal communities in roots of winter wheat (Triticum aestivum) growing in conventional and organic farming systems. Direct and nested polymerase chain reaction (PCR) amplifications spanning the internal transcribed spacer (ITS) region of the rDNA from pooled fine root samples were performed with two different sets of fungal specific primers. Fungal identification was carried out through similarity searches against validated reference sequences (RefSeq). The R package 'picante' and FUNGuild were used to analyse fungal community composition and trophic mode, respectively. Either by direct or cloning sequencing, 130 complete ITS sequences were clustered into 39 operational taxonomic units (OTUs) (25 singletons), belonging to the Ascomycota (24), the Basidiomycota (14) and to the Glomeromycota (1). Fungal communities from conventional farming sites are phylogenetically more related than expected by chance. Constrained ordination analysis identified total N, total S and Pcal that had a significant effect on the OTU's abundance and distribution, and a further correlation with the diversity of the co-occurring vegetation could be hypothesised. The functional predictions based on FUNGuild suggested that conventional farming increased the presence of plant pathogenic fungi compared with organic farming. Based on diversity, OTU distribution, nutrition mode and the significant phylogenetic clustering of fungal communities, this study shows that fungal communities differ across sampling sites, depending on agricultural practices. Although it is not fully clear which factors determine the fungal communities, our findings suggest that organic farming systems have a positive effect on fungal communities in winter wheat crops.

On the Evolutionary History of Uleiella chilensis, a Smut Fungus Parasite of Araucaria araucana in South America: Uleiellales ord. nov. in Ustilaginomycetes.

The evolutionary history, divergence times and phylogenetic relationships of Uleiella chilensis (Ustilaginomycotina, smut fungi) associated with Araucaria araucana were analysed. DNA sequences from multiple gene regions and morphology were analysed and compared to other members of the Basidiomycota to determine the phylogenetic placement of smut fungi on gymnosperms. Divergence time estimates indicate that the majority of smut fungal orders diversified during the Triassic-Jurassic period. However, the origin and relationships of several orders remain uncertain. The most recent common ancestor between Uleiella chilensis and Violaceomyces palustris has been dated to the Lower Cretaceous. Comparisons of divergence time estimates between smut fungi and host plants lead to the hypothesis that the early Ustilaginomycotina had a saprobic lifestyle. As there are only two extant species of Araucaria in South America, each hosting a unique Uleiella species, we suggest that either coevolution or a host shift followed by allopatric speciation are the most likely explanations for the current geographic restriction of Uleiella and its low diversity. Phylogenetic and age estimation analyses, ecology, the unusual life-cycle and the peculiar combination of septal and haustorial characteristics support Uleiella chilensis as a distinct lineage among the Ustilaginomycotina. Here, we describe a new ustilaginomycetous order, the Uleiellales to accommodate Uleiella. Within the Ustilaginomycetes, Uleiellales are sister taxon to the Violaceomycetales.

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.

Phylogenetic relationships in Cortinarius, section Calochroi, inferred from nuclear DNA sequences.

BACKGROUND: Section Calochroi is one of the most species-rich lineages in the genus Cortinarius (Agaricales, Basidiomycota) and is widely distributed across boreo-nemoral areas, with some extensions into meridional zones. Previous phylogenetic studies of Calochroi (incl. section Fulvi) have been geographically restricted; therefore, phylogenetic and biogeographic relationships within this lineage at a global scale have been largely unknown. In this study, we obtained DNA sequences from a nearly complete taxon sampling of known species from Europe, Central America and North America. We inferred intra- and interspecific phylogenetic relationships as well as major morphological evolutionary trends within section Calochroi based on 576 ITS sequences, 230 ITS + 5.8S + D1/D2 sequences, and a combined dataset of ITS + 5.8S + D1/D2 and RPB1 sequences of a representative subsampling of 58 species. RESULTS: More than 100 species were identified by integrating DNA sequences with morphological, macrochemical and ecological data. Cortinarius section Calochroi was consistently resolved with high branch support into at least seven major lineages: Calochroi, Caroviolacei, Dibaphi, Elegantiores, Napi, Pseudoglaucopodes and Splendentes; whereas Rufoolivacei and Sulfurini appeared polyphyletic. A close relationship between Dibaphi, Elegantiores, Napi and Splendentes was consistently supported. Combinations of specific morphological, pigmentation and molecular characters appear useful in circumscribing clades. CONCLUSION: Our analyses demonstrate that Calochroi is an exclusively northern hemispheric lineage, where species follow their host trees throughout their natural ranges within and across continents. Results of this study contribute substantially to defining European species in this group and will help to either identify or to name new species occurring across the northern hemisphere. Major groupings are in partial agreement with earlier morphology-based and molecular phylogenetic hypotheses, but some relationships were unexpected, based on external morphology. In such cases, their true affinities appear to have been obscured by the repeated appearance of similar features among distantly related species. Therefore, further taxonomic studies are needed to evaluate the consistency of species concepts and interpretations of morphological features in a more global context. Reconstruction of ancestral states yielded two major evolutionary trends within section Calochroi: (1) the development of bright pigments evolved independently multiple times, and (2) the evolution of abruptly marginate to flattened stipe bulbs represents an autapomorphy of the Calochroi clade.

Diverse tulasnelloid fungi form mycorrhizas with epiphytic orchids in an Andean cloud forest.

The mycorrhizal state of epiphytic orchids has been controversially discussed, and the state and mycobionts of the pleurothallid orchids, occurring abundantly and with a high number of species on stems of trees in the Andean cloud forest, were unknown. Root samples of 77 adult individuals of the epiphytic orchids Stelis hallii, S. superbiens, S. concinna and Pleurothallis lilijae were collected in a tropical mountain rainforest of southern Ecuador. Ultrastructural evidence of symbiotic interaction was combined with molecular sequencing of fungi directly from the mycorrhizas and isolation of mycobionts. Ultrastructural analyses displayed vital orchid mycorrhizas formed by fungi with an imperforate parenthesome and cell wall slime bodies typical for the genus Tulasnella. Three different Tulasnella isolates were obtained in pure culture. Phylogenetic analysis of nuclear rDNA sequences from coding regions of the ribosomal large subunit (nucLSU) and the 5.8S subunit, including parts of the internal transcribed spacers, obtained directly from the roots and from the fungal isolates, yielded seven distinct Tulasnella clades. Tulasnella mycobionts in Stelis concinna were restricted to two Tulasnella sequence types while the other orchids were associated with up to six Tulasnella sequence types. All Tulasnella sequences are new to science and distinct from known sequences of mycobionts of terrestrial orchids. The results indicate that tulasnelloid fungi, adapted to the conditions on tree stems, might be important for orchid growth and maintenance in the Andean cloud forest.

Morphological and molecular phylogenetic studies in South American Cortinarius species.

Thirty South American species of Cortinarius belonging to the subgenera Telamonia, Dermocybe, Myxacium, Phlegmacium, and Cystogenes were studied using an integrated approach that included morphological, anatomical, and ultrastructural data, and also molecular phylogenetic analysis of nuclear rDNA sequences. The micromorphology of the basidiomes was studied by light microscopy, and the principal structures were illustrated by line drawings. Basidiospore ornamentation was studied by scanning electron microscopy (SEM). Nuclear internal transcribed spacers (ITS, including the 5.8S gene) and the rDNA coding for the D1/D2 domains of the large ribosomal subunit (LSU) were sequenced and analysed using a Bayesian Markov chain Monte Carlo method to estimate phylogenetic relationships between the studied Cortinarius species. Morphology and anatomy of the pileus surface and basidiome pigmentation appeared to be the most useful characters to delimit some natural groups, whereas microcharacters related to the structure of pileus context, hymenophoral and stipe trama were of little taxonomic value. Basidiospore morphology and cheilocystidia seem to be taxonomically relevant at the species level. The following five infrageneric groups were supported by the morphological, chemical and molecular data: (1) Telamonia characterized by wide hyaline hyphae of the veil and by small basidiomes; (2) Dermocybe spp. with an epicutis as the most external layer of the pileus, and skyrin and hypericin pigments; (3) Dermocybe spp. with a thin viscid layer on the pileus, and endocrocin and dermolutein pigments; (4) Phlegmacium spp. characterized by a long and radicating stipe; and (5) Phlegmacium spp. that overlap in some macrocharacters with Telamonia species. Our analyses suggest that classification concepts based mainly on macromorphological characters are likely to lead to artificial grouping, whereas certain microscopical and chemical characters seem to be useful in constructing a more natural classification system for Cortinarius.