The present study focuses on potential agents of chromoblastomycosis and other endemic diseases in the state of Paraná, Southern Brazil. Using a highly selective protocol for chaetothyrialean black yeasts and relatives, environmental samples from the living area of symptomatic patients were analysed. Additional strains were isolated from creosote-treated wood and hydrocarbon-polluted environments, as such polluted sites have been supposed to enhance black yeast prevalence. Isolates showed morphologies compatible with the traditional etiological agents of chromoblastomycosis, e.g. Fonsecaea pedrosoi and Phialophora verrucosa, and of agents of subcutaneous or systemic infections like Cladophialophora bantiana and Exophiala jeanselmei. Some agents of mild disease were indeed encountered. However, molecular analysis proved that most environmental strains differed from known etiologic agents of pronounced disease syndromes: they belonged to the same order, but mostly were undescribed species. Agents of chromoblastomycosis and systemic disease thus far are prevalent on the human host. The hydrocarbon-polluted environments yielded yet another spectrum of chaetothyrialean fungi. These observations are of great relevance because they allow us to distinguish between categories of opportunists, indicating possible differences in pathogenicity and virulence.

The genus Fonsecaea is revised on the basis of ribosomal DNA internal transcribed spacer (ITS) sequence data. Two species are recognized, F. pedrosoi and the new defined F. monophora. The distinction between these species does not correspond with the classical distinction of F. pedrosoi and F. compacta. The latter appears to be no more than a morphological variant. Both species recognized in this study are agents of human chromoblastomycosis; however, in F. pedrosoi a strict association with this disease is noted, while F. monophora is a more general opportunist. Subspecific randomly amplified polymorphic DNA (RAPD) typing revealed a high degree of strain diversity, although clonal reproduction is also likely to occur. Most strains with Fonsecaea-like morphology isolated from environments to which symptomatic human patients were exposed were found to be more closely related to species of Cladophialophora than to Fonsecaea.

Black yeast members of the Herpotrichiellaceae present a complex ecological behavior: They are often isolated from rather extreme environments polluted with aromatic hydrocarbons, while they are also regularly involved in human opportunistic infections. A selective technique to promote the in vitro growth of herpotrichiellaceous fungi was applied to investigate their ecophysiology. Samples from natural ecological niches and man-made environments that might contain black yeasts were enriched on an inert solid support at low humidity and under a controlled atmosphere rich in volatile aromatic hydrocarbons. Benzene, toluene, and xylene were provided separately as the sole carbon and energy source via the gas phase. The assayed isolation protocol was highly specific toward mesophilic Exophiala species (70 strains of this genus out of 71 isolates). Those were obtained predominantly from creosote-treated railway ties (53 strains), but isolates were also found on wild berries (11 strains) and in guano-rich soil samples (six strains). Most of the isolates were obtained on toluene (43 strains), but enrichments on xylene and benzene also yielded herpotrichiellaceous fungi (17 and 10 isolates, respectively). Based upon morphological characterizations and DNA sequences of the full internal transcriber spacers (ITS) and the 8.5S rRNA genes, the majority of the obtained isolates were affiliated to the recently described species Exophiala xenobiotica (32 strains) and Exophiala bergeri (nine strains). Members of two other phylogenetic groups (24 and two strains, respectively) somewhat related to E. bergeri were also found, and a last group (three strains) corresponded to an undescribed Exophiala species.

Fonsecaea and Cladophialophora are genera of black yeast-like fungi harboring agents of a mutilating implantation disease in humans, along with strictly environmental species. The current hypothesis suggests that those species reside in somewhat adverse microhabitats, and pathogenic siblings share virulence factors enabling survival in mammal tissue after coincidental inoculation driven by pathogenic adaptation. A comparative genomic analysis of environmental and pathogenic siblings of Fonsecaea and Cladophialophora was undertaken, including de novo assembly of F. erecta from plant material. The genome size of Fonsecaea species varied between 33.39 and 35.23 Mb, and the core genomes of those species comprises almost 70% of the genes. Expansions of protein domains such as glyoxalases and peptidases suggested ability for pathogenicity in clinical agents, while the use of nitrogen and degradation of phenolic compounds was enriched in environmental species. The similarity of carbohydrate-active vs. protein-degrading enzymes associated with the occurrence of virulence factors suggested a general tolerance to extreme conditions, which might explain the opportunistic tendency of Fonsecaea sibling species. Virulence was tested in the Galleria mellonella model and immunological assays were performed in order to support this hypothesis. Larvae infected by environmental F. erecta had a lower survival. Fungal macrophage murine co-culture showed that F. erecta induced high levels of TNF-α contributing to macrophage activation that could increase the ability to control intracellular fungal growth although hyphal death were not observed, suggesting a higher level of extremotolerance of environmental species.

The oil flotation isolation technique has been successfully applied to recover chaetothyrialean black yeasts and relatives from the environment. The selective mechanisms playing a role in isolation are unknown. The fungi concerned are supposed to occupy specialized microniches in nature, taking advantage of (1) oligotrophism. Mineral oil as a main selective agent may be based on (2) hydrophobicity or on (3) assimilation. All three hypotheses are tested in this paper. Results show that cell wall hydrophobicity is unlikely to be a selective factor. Incubation under poor nutrient conditions provides competitive advantage for black yeasts, especially for Exophiala strains, which are subsequently enriched by mineral oil which enhances growth in this group of fungi. Incubation under mineral media and mineral oil can be used as selective factor.

Ochroconis globalis, a novel species of the melanized genus Ochroconis (Sympoventuriaceae, Venturiales), is described and illustrated and distinguished phenotypically and molecularly from existing ochroconis-like species. Phylogenetic analyses using nuclear ribosomal DNA genes (nuSSU, ITS, nuLSU) and coding gene fragments (ACT1, BT2, TEF1) revealed clustering of all strains as a monophyletic clade which was well separated from known Ochroconis species. Most strains of the new species were obtained from human-made environments, while the natural habitat of the species remains enigmatic. The new combination Ochroconis musae is introduced for one of the most commonly encountered Ochroconis species, and a phenotypic key to all species is provided.

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Fungus-growing ants of the genus Atta are known for their leaf-cutting habit, a lifestyle they have maintained since their 50-million-year-old co-evolution with a mutualistic fungus, cultivated as food. Recent studies have highlighted that, in addition to the mutualistic fungus, nests of ants harbor a great diversity of microbial communities. Such microorganisms include the dematiaceous fungi, which are characterized by their melanized cell walls. In order to contribute to the knowledge of fungal ecology, as well as opportunistic strains that may be dispersed by these social insects, we isolated and identified fungi carried by gynes of Atta capiguara and Atta laevigata, collected from colonies located in Fazenda Santana, Botucatu (São Paulo, Brazil). The isolation was carried out using the oil flotation technique, which is suitable for the growth of black fungi. Inoculated plates were incubated at 25 and 35 °C until black cultures were visible (20-45 days). Isolates were identified based on microscopic and molecular characteristics. Some isolated genera were: Cladophialophora, Cladosporium, Exophiala, Ochroconis, Phaeococcomyces, Phialophora and Penidiella. Hyaline species were also found. The results obtained from this work showed that leaf-cutting gynes may contribute to the dispersal of opportunistic dematiaceous fungi. It is suggested that more attention should be paid to this still unexplored subject.